Methods and compositions for the modulation of chorismate synthase and chorismate mutase expression or activity in plants

ABSTRACT

The present inventors have discovered that chorismate mutase and chorismate synthase are essential for plant growth. Specifically, the inhibition of chorismate mutase or chorismate synthase gene expression in plant seedlings results in severe chlorosis, reduced growth and developmental abnormalities. The inventors have proven that chorismate synthase and chorismate mutase can be used as targets for the identification of herbicides. Thus, the invention provides methods for the identification of chemicals that modulate chorismate synthase and chorismate mutase biochemical reactions. The methods of the invention are useful for the identification of herbicides and for the inhibition of plant growth and development.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 09/610,040, filed on Jul. 5, 2000, now U.S. Pat. No. 6,465,217.

FIELD OF THE INVENTION

The invention relates generally to plant molecular biology. Inparticular, the invention relates to compositions and methods for theregulation of plant growth and development through the modulation ofeither chorismate synthase or chorismate mutase gene expression oractivity.

BACKGROUND OF THE INVENTION

Chorismate is an essential substrate for the synthesis ofp-aminobenzoate, folate, ubiquinone and the aromatic amino acidstryptophan, phenylalanine and tyrosine. Chorismate is produced in theseventh step of the shikimate pathway. This pathway has been describedin numerous publications. See, for example, Shikimic Acid: Metabolismand Metabolites, John Wiley & Sons, Winchester, UK, 1993. The first foursteps of the shikimate biosynthetic pathway lead to the production ofshikimate. Shikimate is then converted to chorismate in next three stepsof the pathway. First, shikimate is converted to shikimate-5-phosphateby shikimate kinase. Next, 3-enolpyruvateshikimate 5-phosphate synthaseconverts shikimate-5-phosphate to 5-enolpyruvylshikimate 3-phosphate,which is then converted to chorismate by the enzyme chrosimate synthase.

The pathway leading to aromatic amino acid synthesis branches atchorismate. One branch leads to the synthesis of tryptophan. The otherbranch leads to the synthesis of phenylalanine and tyrosine. Thus,chorismate is the last common intermediate in the synthesis oftryptophan, phenylalanine and tyrosine.

In the branch leading to phenylalanine and tyrosine synthesis,chorismate is converted to prephenate by the enzyme chorismate mutase.Prephenate is the last common intermediate for biosyntheses ofphenylalanine and tyrosine by two independent pathways that are presentin both eukaryotes and prokaryotes.

Three isozymes of chorismate mutase, CM-1, CM-2 and CM-3 have been foundin plants. Mobley et al. (1999) Gene 240:115-123. CM-1 and CM-3 areplastidic, while CM-2 is cytosolic. In Arabidopsis thaliana, CM-1 has53% amino acid similarity with CM-2 and 68% amino acid similarity withCM-3.

The conversion of shikimate-5-phosphate to 5-enolpyruvylshikimate3-phosphate is blocked by the commercially successful herbicide Roundup™(glyphosate). Accordingly, the shikimate pathway has been considered anattractive target for herbicides (PCT publication WO 00/05353, thecontents of which are incorporated by reference, and Roberts et al.(1998) Nature 393:801-805). However, while it has been suggested thatthat chorismate synthase could be a candidate for a herbicide target(Bomemann et al. (1985) J Biol Chem 270:228111-22815), previous studieshave not been able to ascertain whether chorismate synthase orchorismate mutase are essential for plant growth, which is a keyparameter for determining potential herbicide targets. Nor are there anyherbicides that are known to act by modifying the activity of either ofthese enzymes. Thus, it is necessary to determine whether such enzymesare critical to plant growth, before they can be considered usefultargets in assays for the identification of herbicides and herbicidecandidates.

SUMMARY OF THE INVENTION

The present inventors have discovered that chorismate mutase andchorismate synthase are essential for plant growth. Specifically, theinhibition of chorismate mutase or chorismate synthase gene expressionin plant seedlings results in severe chlorosis, reduced growth anddevelopmental abnormalities. Thus, in one aspect, the present inventionprovides compositions for the modulation of plant growth or developmentcomprising chorismate synthase and chorismate mutase antisense and sensepolynucleotides, dsRNA and ribozymes, and related expression cassettesand vectors. The compositions of the invention are particularly usefulfor the modulation and inhibition of plant growth. The invention furtherprovides plants, plant cells, and seeds containing the polynucleotidesof the invention.

The inventors have proven that chorismate synthase and chorismate mutasecan be used as targets for the identification of herbicides. Thus, thepresent invention also provides methods for the identification ofchemicals that modulate chorismate synthase and chorismate mutasebiochemical reactions. The methods of the invention are useful for theidentification of herbicides and for the inhibition of plant growth anddevelopment. In addition, the methods of the invention are useful forthe identification of compounds that stimulate the expression orfunction of chorismate synthase or chorismate mutase expression orfunction. Such compounds can be used to promote or manipulate plantgrowth and development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the driver expression cassette.

FIG. 2 is a diagram of the target expression cassette.

FIG. 3 shows activation of the target antisense expression cassette bythe driver transactivation factor.

FIG. 4 is a digital image showing the effect of chorismate synthaseantisense expression on Arabidopsis thaliana seedlings.

FIG. 5 is a digital photograph image showing the effect of chorismatemutase-1 antisense expression on A. thaliana seedlings.

DETAILED DESCRIPTION OF THE INVENITON

Definitions

The term “antisense”, for the purposes of the invention, refers to anucleic acid comprising a polynucleotide which is sufficientlycomplementary to all or a portion of a chorismate synthase gene,chorismate mutase gene, primary transcripts or processed mRNAs derivedfrom chorismate synthase or chorismate mutase genes, so as to interferewith expression of the endogenous chorismate synthase or chorismatemutase genes.

The term “binding” refers to a noncovalent interaction that holds twomolecules together. For example, two such molecules could be an enzymeand an inhibitor of that enzyme. Noncovalent interactions includehydrogen bonding, ionic interactions among charged groups, van der Waalsinteractions and hydrophobic interactions among nonpolar groups. One ormore of these interactions can mediate the binding of two molecules toeach other.

As used herein, the term “chorismate” is synonymous with “chorismicacid”. The structure is shown as entry number 2274 in the Merck Index,Twelfth Edition, Budavari et al., Eds., Merck Research Laboratories,Whitehouse Station, N.J., 1996.

“Complementary” polynucleotides are those which are capable ofbase-pairing according to the standard Watson-Crick complementarityrules. Specifically, purines will base pair with pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Two polynucleotides may hybridize toeach other if they are complementary to each other, or if each has atleast one region that is substantially complementary to the other.

The structure of “5-enolpyruvylshikimate 3-phosphate” is shown inMacheroux et al. (1996) J Biol Chem 271:25850-25858.

The term “herbicide”, as used herein, refers to a chemical that may beused to kill or suppress the growth of at least one plant, plant cell,plant tissue or seed.

By “herbicidally effective amount” is meant an amount of a chemical orcomposition sufficient to kill a plant or decrease plant growth and/orviability by at least 10%. More preferably, the growth or viability willbe decreased by 25%, 50%, 75%, 80%, 90% or more.

For the purposes of the invention, “high stringency hybridizationconditions” refers to hybridization in 50% formamide, 1 M NaCL, 1% SDSat 37° C., and a final wash in 0.1×SSC at 60° C. Methods for nucleicacid hybridizations are described in Meinkoth and Wahl (1984) AnalBiochem 138:267-284; Current Protocols in Molecular Biology, Chapter 2,Ausubel et al. Eds., Greene Publishing and Wiley-Interscience, New York,1995; and Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2,Elsevier, N.Y., 1993.

The term “inhibitor”, as used herein, refers to a chemical substancethat inactivates the enzymatic activity of chorismate synthase orchorismate mutase. The inhibitor may function by interacting directlywith the enzyme, a cofactor of the enzyme, the substrate of the enzyme,or any combination thereof.

A polynucleotide may be “introduced” into a plant cell by any means,including transfection, transformation or transduction, electroporation,particle bombardment, Agroinfection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the plant chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

For the purposes of the invention, an “isolated polynucleotide” is apolynucleotide that is substantially free of the nucleic acid sequencesthat normally flank the polynucleotide in it naturally occurringreplicon. For example, a cloned polynucleotide is considered isolated.Alternatively, a polynucleotide is considered isolated if it has beenaltered by human intervention, or placed in a locus or location that isnot its natural site, or if it is introduced into cell by agroinfection.

By “male tissue” is meant the tissues of a plant that are directlyinvolved or supportive of the reproduction of the male gametes. Suchtissues include pollen tapetum, anther, tassel, pollen mother cells andmicrospores. A “male tissue-preferred” or “male tissue-specific”promoter will be expressed predominantly in one or more male tissues. Itis possible that a male tissue preferred promoter will be expressed innon-male tissues, however, expression will usually be at a lower levelthan in male tissues.

As used herein, “nucleic acid” and “polynucleotide” refers to RNA or DNAthat is linear or branched, single or double stranded, or a hybridthereof. The term also encompasses RNA/DNA hybrids. Less common bases,such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine andothers can also be used for antisense, dsRNA and ribozyme pairing. Forexample, polynucleotides which contain C-5 propyne analogues of uridineand cytidine have been shown to bind RNA with high affinity and to bepotent antisense inhibitors of gene expression. Other modifications,such as modifications to the phosphodiester backbone, or the 2′-hydroxyin the ribose sugar group of the RNA can also be made. The antisensepolynucleotides and ribozymes can consist entirely of ribonucleotides,or can contain mixed ribonucleotides deoxyribonucleotides. Thepolynucleotides of the invention may be produced by any means, includinggenomic preparations, cDNA preparations, in vitro synthesis, RT-PCR andin vitro or in vivo transcription.

By “operably linked” is meant that a polynucleotide is functionallylinked to a promoter, so that the transcription of the polynucleotidecan be initiated from the promoter.

The “percent sequence identity” between two polynucleotide or twopolypeptide sequences is determined according to the either the BLASTprogram (Basic Local Alignment Search Tool; Altschul and Gish (1996)Meth Enzymol 266:460-480 and Altschul (1990) J Mol Biol 215:403-410) inthe Wisconsin Genetics Software Package (Devererreux et al. (1984) NuclAcid Res 12:387), Genetics Computer Group (GCG), Madison, Wis. (NCBI,Version 2.0.11, default settings) or using Smith Waterman Alignment(Smith and Waterman (1981) Adv Appl Math 2:482) as incorporated intoGeneMatcher Plus™ (Paracel, Inc.,http://www.paracel.com/html/genematcher.html; using the default settingsand the version current at the time of filing). It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

“Plant” refers to whole plants, plant organs and tissues (e.g., stems,roots, ovules, stamens, leaves, embryos, meristematic regions, callustissue, gametophytes, sporophytes, pollen, microspores and the like)seeds, plant cells and the progeny thereof.

By “plant chorismate mutase RNA” is meant a primary transcript orprocessed mRNA derived from any plant chorismate mutase gene.

By “plant chorismate synthase RNA” is meant a primary transcript orprocessed mRNA derived from any plant chorismate synthase gene.

By “polypeptide” is meant a chain of at least four amino acids joined bypeptide bonds. The chain may be linear, branched, circular orcombinations thereof. The polypeptides may contain amino acid analogsand other modifications, including, but not limited to glycosylated orphosphorylated residues.

As used herein, the term “prephenate” is synonymous with “prephenicacid”. The structure is shown as compound number 7920 in the MerckIndex, Twelfth Edition, Budavari et al., Eds., Merck ResearchLaboratories, Whitehouse Station, N.J., 1996.

As used herein, the term “probe” refers to can have no more than anadditional 10 nucleic acid residues at either end of a polynucleotidehaving a defined sequence.

For the purposes of the invention, “recombinant polynucleotide” refersto a polynucleotide that has been altered, rearranged or modified bygenetic engineering. Examples include any cloned polynucleotide, andpolynucleotides that are linked or joined to heterologous sequences. Twopolynucleotide sequences are heterologous if they are not naturallyfound joined together. The term recombinant does not refer toalterations to polynucleotides that result from naturally occurringevents, such as spontaneous mutations.

By “ribozyme” is meant a catalytic RNA-based enzyme capable of targetingand cleaving particular base sequences in both DNA and RNA. Ribozymescomprise a polynucleotide sequence that is complementary to a portion ofa target nucleic acid and a catalytic region that cleaves the targetnucleic acid. Ribozymes can be designed that specifically pair with andinactivate a target RNA by catalytically cleaving the RNA at a targetedphosphodiester bond. Methods for making and using ribozymes are known tothose skilled in the art. See, for example, U.S. Pat. Nos. 6,025,167;5,773,260 and 5,496,698, the contents of which are incorporated byreference, and in Haseloff and Gerlach (1988) Nature 334:586-591.

The term “specific binding” refers to an interaction between chorismatesynthase or chorismate mutase and a molecule or compound, wherein theinteraction is dependent upon the primary amino acid sequence or theconformation of chorismate synthase or chorismate mutase.

“Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,agroinfection, electroporation, particle bombardment, and the like. Thisprocess may result in transient or stable (constitutive or regulated)expression of the transformed polynucleotide. By “stably transformed” ismeant that the sequence of interest is integrated into a replicon in thecell, such as a chromosome or episome. Transformed cells, tissues andplants encompass not only the end product of a transformation process,but also the progeny thereof which retain the polynucleotide ofinterest.

For the purposes of the invention, “transgenic” refers to any plant,plant cell, callus, plant tissue or plant part, that contains all orpart of at least one recombinant polynucleotide. In many cases, all orpart of the recombinant polynucleotide is stably integrated into achromosome or stable extra-chromosomal element, so that it is passed onto successive generations.

Embodiments of the Invention

The present inventors have discovered that inhibition of chorismatesynthase or chorismate mutase gene expression strongly inhibits thegrowth and development of plant seedlings. Thus, the inventors are thefirst to show that chorismate synthase and chorismate mutase are targetsfor herbicides.

Accordingly, the invention provides methods for identifying compoundsthat modulate chorismate synthase or chorismate mutase gene expressionor activity. Such methods include ligand binding assays, assays forenzyme activity and assays for chorismate synthase and chorismate mutasegene expression. The compounds identified by the methods of theinvention are useful for the modulation of plant growth and development.

Any compound that is a ligand for chorismate synthase, other than itssubstrate, 5-enolpyruvylshikimate 3-phosphate, may have herbicidalactivity. For the purposes of the invention, “ligand” refers to amolecule that will bind to a site on a polypeptide. Thus, in oneembodiment, the invention provides a method for identifying a compoundas a candidate for a herbicide, comprising:

a) combining said compound with at least one polypeptide selected fromthe group consisting of: a plant chorismate synthase, a polypeptidecomprising at least ten consecutive amino acids of a plant chorismatesynthase, a polypeptide having at least 85% sequence identity with aplant chorismate synthase, and a polypeptide having at least 80%sequence identity with a plant chorismate synthase and at least 50% ofthe activity thereof; and

b) detecting the presence and/or absence of binding between saidcompound and said polypeptide;

wherein binding indicates that said compound is a candidate for aherbicide.

Similarly, any compound that is a ligand for chorismate mutase, otherthan its substrate, chorismate, may have herbicidal activity. Thus, inanother embodiment, the invention provides a method for identifying acompound as a candidate for a herbicide, comprising:

a) combining said compound with at least one polypeptide selected fromthe group consisting of: a plant chorismate mutase, a polypeptidecomprising at least ten consecutive amino acids of a plant chorismatemutase, a polypeptide having at least 85% sequence identity with a plantchorismate mutase and a polypeptide having at least 80% sequenceidentity with a plant chorismate mutase and at least 50% of the activitythereof; and

b) detecting the presence and/or absence of binding between saidcompound and said polypeptide;

wherein binding indicates that said compound is a candidate for aherbicide.

By “plant chorismate synthase” and “plant chorismate mutase” is meantthe polypeptide corresponding to chorismate synthase or chorismatemutase, respectively, that can be found in at least one plant. Thechorismate synthase may be from any plant, including both monocots anddicots. In various embodiments, the chorismate synthase or chorismatemutase is from barnyard grass (Echinochloa crus-galli), crabgrass(Digitaria sanguinalis), green foxtail (Setana viridis), perennialryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade(Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf(Abutilon theophrasti), common lambsquarters (Chenopodium album L.),Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia,Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthusretroflexus, Sida spinosa, Xanthium strumarium and the like. In oneembodiment, the chorismate synthase or chorismate mutase is anArabidopsis chorismate synthase, chorismate mutase-1, or chorismatemutase-3.

Fragments of a plant chorismate synthase or chorismate mutase may beused in the methods of the invention. The fragments comprise at least 5consecutive amino acids of a plant chorismate synthase or a plantchorismate mutase. Preferably, the fragment comprises at least 6, 7, 8,9, 10, 15, 20, 25, 30, 35 or at least 40 consecutive amino acidsresidues of a plant chorismate synthase or chorismate mutase.

Polypeptides having at least 80% sequence identity with a plantchorismate mutase or a plant chorismate synthase are also useful in themethods of the invention. Preferably, the sequence identity is at least85%, more preferably the identity is at least 90%, most preferably thesequence identity is at least 95%.

In addition, it is preferred that the polypeptide has at least 50% ofthe activity of a plant chorismate synthase or chorismate mutase. Morepreferably, the polypeptide has at least 60%, at least 70%, at least 80%or at least 90% of the activity of a plant chorismate synthase orchorismate mutase.

Preferably, the activity of the polypeptide is compared to the activityof the Arabidopsis chorismate synthase polypeptide or an Arabidopsischorismate mutase-1 polypeptide. For the purposes of the invention,chorismate synthase activity refers to the ability to convert5-enolpyruvateshikimate 3-phosphate to chorismate. Chorismate mutaseactivity refers to the ability to convert chorismate to prephenate.Methods for measuring chorismate synthase and chorismate mutase activityare known in the art. Preferably, chorismate synthase activity ismeasured according to the method described in Henstrand et al. (1995) JBiol Chem 270:20447-20452. In another preferred embodiment, chorismatemutase activity is measured according to the method described inKrappmann et al. (1999) J Biol Chem 274:22275-22282.

Any technique for detecting the binding of a ligand to its target may beused in the methods of the invention. Preferably, the ligand and targetare combined in a buffer. Polypeptides and proteins that can reducenon-specific binding, such as BSA or protein extracts from cells that donot produce the target, may be included in binding assay.

Many methods for detecting the binding of a ligand to its target areknown in the art, and include, but are not limited to the detection ofan immobilized ligand-target complex or the detection of a change in theproperties of a target when it is bound to a ligand. For example, in oneembodiment, an array of immobilized candidate ligands is provided. Theimmobilized ligands are contacted with a plant chorismate synthaseprotein or a fragment or variant thereof, the unbound protein is removedand the bound chorismate synthase is detected. In a preferredembodiment, bound chorismate synthase is detected using a labeledbinding partner, such as a labeled antibody. In a variation of thisassay, chorismate synthase is labeled prior to contacting theimmobilized candidate ligands. Preferred labels include fluorescent orradioactive moieties. Preferred detection methods include fluorescencecorrelation spectroscopy (FCS) and FCS-related confocal nanofluorimetricmethods. See http://www.evotec.de/technology. The same methods areapplicable to the detection of binding of a candidate ligand tochorismate mutase.

Once a compound is identified as a candidate for a herbicide, it can betested for the ability to inhibit or otherwise modulate chorismatemutase or chorismate synthase enzyme activity. The compounds can betested using either in vitro or cell based enzyme assays. Alternatively,a compound can be tested by applying it directly to a plant or plantcell, or expressing it therein, and monitoring the plant or plant cellfor changes or decreases in growth, development, viability oralterations in gene expression.

Thus, in one embodiment, the invention provides a method for determiningwhether a compound identified as a herbicide candidate by an abovemethod has herbicidal activity, comprising: contacting a plant or plantcells with said herbicide candidate and detecting the presence orabsence of a decrease in the growth or viability of said plant or plantcells.

By decrease in growth, is meant that the herbicide candidate causes atleast a 10% decrease in the growth of the plant or plant cells, ascompared to the growth of the plants or plant cells in the absence ofthe herbicide candidate. By a decrease in viability is meant that atleast 10% of the plants cells, or portion of the plant contacted withthe herbicide candidate are nonviable. Preferably, the growth orviability will be at decreased by at least 20%. More preferably, thegrowth or viability will be decreased by at least 50%, 75% or at least90% or more. Methods for measuring plant growth and cell viability areknown to those skilled in the art. Clearly, it is possible that acandidate compound may have herbicidal activity only for certain plantsor certain plant species.

The ability of a compound to inhibit or modulate chorismate synthase orchorismate mutase activity can be detected using in vitro enzymaticassays in which the disappearance of a substrate or the appearance of aproduct is detected. Chorismate synthase converts 5-enolpyruvylshikimate3-phosphate to chorismate. Chorismate mutase converts chorismate toprephenate. Methods for detection of these substrates and products, suchas mass spectroscopy and reverse phase HPLC, are known to those skilledin the art.

Thus, the invention provides a method for identifying a compound as acandidate for a herbicide, comprising:

a) combining 5-enolpyruvylshikimate 3-phosphate and chorismate synthaseunder reaction conditions suitable for chorismate synthase activity;

b) combining 5-enolpyruvylshikimate 3-phosphate, chorismate synthase andsaid compound under the reaction conditions of step (a);

c) detecting the amount of 5-enolpyruvyishikimate 3-phosphate and/orchorismate in steps (a) and (b).

In this method, if a candidate compound inhibits chorismate synthaseactivity, a higher concentration of the precursor(5-enolpyruvylshikimate 3-phosphate) and a lower level of the product(chorismate) will be detected in the presence of the candidate compound(step b) than in the absence of the compound (step a).

Preferably the chorismate synthase is a plant chorismate synthase.

Enzymatically active fragments of a plant chorismate synthase are alsouseful in the methods of the invention. For example, a polypeptidecomprising at least ten consecutive amino acid residues of a plantchorismate synthase may be used in the methods of the invention. Inaddition, a polypeptide having at least 80%, 85%, 90%, 95%, 98% or atleast 99% sequence identity with a plant chorismate synthase may be usedin the methods of the invention. Also, polypeptides having at least 80%sequence identity with at least 15 consecutive amino acid residues of aplant chorismate synthase are also useful in the methods of theinvention. Preferably, the polypeptide has at least 80% sequenceidentity with a plant chorismate synthase and at least 50%, 75%, 90% orat least 95% of the activity thereof.

Thus, the invention provides a method for identifying a compound as acandidate for a herbicide, comprising:

a) combining, under reaction conditions suitable for chorismate synthaseactivity, 5-enolpyruvylshikimate 3-phosphate and a polypeptide selectedfrom the group consisting of: a polypeptide having at least 85% sequenceidentity with a plant chorismate synthase, a polypeptide having at least80% sequence identity with a plant chorismate synthase and at least 50%of the activity thereof, and a polypeptide comprising at least 10consecutive amino acids of a plant chorismate synthase;

b) combining 5-enolpyruvylshikimate 3-phosphate, said polypeptide andsaid compound under the reaction conditions of step (a);

c) detecting the amount of 5-enolpyruvylshikimate 3-phosphate and/orchorismate in steps (a) and (b).

Again, if a candidate compound inhibits chorismate synthase activity, ahigher concentration of the precursor (5-enolpyruvylshikimate3-phosphate) and a lower level of the product (chorismate) will bedetected in the presence of the candidate compound (step b) than in theabsence of the compound (step a).

These methods are useful for identifying compounds that inhibitchorismate synthase activity. Other assays for chorismate synthaseactivity are known in the art. See for example, Ramjee et al. (1994)Anal Biochem 220:137-141; and Macheroux et al. (1996) J Biol Chem271:25850-25858. For example, in one assay, chorismate synthase activityis measured by monitoring the appearance of chorismate at 275 nm at 30°C. in a reaction volume of 0.5 ml containing triethanolamine-HCL, pH8.0, 50 mM KCl, 2.5 mM MgCl₂, 200 μM NADPH, 10 μM FMN and 80 μM5-enolpyruvyishikimate 3-phosphate (EPSP). Henstrand et al. (1991) JBiol Chem 270:20447-20452.

Similar methods can be used to identify inhibitors of chorismate mutaseactivity. Chorismate mutase converts chorismate to prephenate. Thus, inanother embodiment, the invention provides a method for identifying acompound as a candidate for a herbicide, comprising:

a) combining chorismate and chorismate mutase under reaction conditionssuitable for chorismate mutase activity;

b) combining chorismate, chorismate mutase and said compound under thereaction conditions of step (a);

c) detecting the amount of chorismate and/or prephenate in steps (a) and(b).

If a candidate compound inhibits chorismate mutase activity, a higherconcentration of the precursor (chorismate) and a lower level of theproduct (prephenate) will be detected in the presence of the compound(step b) than in the absence of the compound (step a).

Preferably the chorismate mutase is a plant chorismate mutase. Morepreferably, the chorismate mutase is a plant chorismate mutase-1 orplant chorismate mutase-3. In one embodiment, a polypeptide comprisingat least ten consecutive amino acid residues of a plant chorismatemutase may be used in the methods of the invention. In addition, apolypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99%sequence identity with a plant chorismate mutase may be used in themethods of the invention. Preferably, the polypeptide has at least 80%sequence identity with a plant chorismate mutase and at least 50%, 75%,90% or at least 95% of the activity thereof.

Thus, in another aspect, the invention provides a method for identifyinga compound as a candidate for a herbicide, comprising:

a) combining, under reaction conditions suitable for chorismate mutaseactivity, chorismate and a polypeptide selected from the groupconsisting of: a polypeptide having at least 85% sequence identity witha plant chorismate mutase, a polypeptide having at least 80% sequenceidentity with a plant chorismate mutase and at least 50% of the activitythereof, and a polypeptide comprising at least 10 consecutive aminoacids of a plant chorismate mutase;

b) combining chorismate, said polypeptide and said compound under thereaction conditions of step (a);

c) detecting the amount of prephenate and/or chorismate in steps (a) and(b).

These methods are useful for identifying compounds that inhibitchorismate mutase activity. Other assays for chorismate mutase are knownin the art. See, for example, Krappmann et al. (1999) J Biol Chem274:22275-22282; Gilchrist and Connelly (1987) Methods in Enzymology142:450-463; Schmidheini et al. (1989) J Bacteriol 171:1245-1253;Gorisch (1978) Anal Biochem 86:764-768; and Gorisch (1987) Methods inEnzymology 142:463.

One assay commonly used to measure chorismate mutase activity is basedon the spectrophotometric estimation of phenylpyruvate obtained by acidconversion of the reaction product of prephenate. Metzenberg andMitchell (1954) Arch Biochem and Biophys 243:374. In this assay,phenylpyruvate is estimated by the absorbance of either theenol-tautomer in alkaline solution (E=17,500 M⁻¹ cm⁻¹ at 320 nm in 1 MNaOH) or the enol-borate complex in either a concentrated solution ofsodium borate and arsenate or phosphate (E=9292 M⁻¹ cm⁻¹ at 300 nm).Alternatively, the disappearance of chorismate may be assayedspectrophotometrically. Nishioka and Woodin (1972) Anal Biochem 45:617.The details of these assays and their advantages and disadvantages aredescribed in Gilchrist and Connelly (1987) Methods in Enzymology142:450-463.

For the in vitro enzymatic assays, chorismate synthase and chorismatemutase proteins may be purified from a plant or may be recombinantlyproduced in and purified from a plant, bacteria, or eukaryotic cellculture. Preferably these proteins are produced using a baculovirusexpression system. Methods for the purification of chorismate synthaseare described in White et al. (1988) Biochem J 251:313-322; Henstrand etal. (1995) J Biol Chem 270:20447-20452; Bornemann et al. (1995) BiochemJ 305:707-710. Methods for the purification of chorismate mutase aredisclosed in Krappmann et al. (1999) J Biol Chem 274:22275-22282;Schmidheini et al. (1990) Biochem 29:3660-3669; and Gilchrist andConnelly (1987) Methods in Enzymology 142:450-463. In a preferredmethod, a 6×histidine tagged chorismate synthase fusion protein or a6×histidine tagged chorismate mutase fusion protein is affinitypurified. See, for example, Grundy et al. (1998) Protein Expr Purif13:61-66; and Hosfield and Lu (1999) Biotechniques 27:58-60.

As an alternative to in vitro assays, the invention also provides plantand plant cell based assays. In one embodiment, the invention provides amethod for identifying a chemical as a candidate for a herbicide,comprising:

a) measuring the expression of chorismate synthase and/or chorismatemutase in a plant or plant cell in the absence of said chemical;

b) contacting a plant or plant cell with said chemical and measuring theexpression of chorismate synthase and/or chorismate mutase in said plantor plant cell;

c) comparing the expression of chorismate synthase and/or chorismatemutase in steps (a) and (b).

A reduction in chorismate synthase or chorismate mutase expressionindicates that the compound is a herbicide candidate. In one embodiment,the plant or plant cell is an Arabidopsis thaliana plant or plant cell.

Expression of chorismate synthase can be measured by detectingchorismate synthase primary transcript or mRNA, chorismate synthasepolypeptide or chorismate synthase enzymatic activity. Similarly,expression of chorismate mutase can be measured by detecting chorismatemutase primary transcript or mRNA, chorismate mutase polypeptide orchorismate mutase enzymatic activity.

Methods for detecting the expression of RNA and proteins are known tothose skilled in the art. See, for example, Current Protocols inMolecular Biology Ausubel et al., eds., Greene Publishing andWiley-Interscience, New York, 1995. The method of detection is notcritical to the invention. Methods for detecting chorismate synthase orchorismate mutase RNA include, but are not limited to amplificationassays such as quantitative PCR, and/or hybridization assays such asNorthern analysis, dot blots, slot blots, in-situ hybridization, bDNAassays and microarray assays.

Methods for detecting protein expression include, but are not limitedto, immunodetection methods such as Western blots, His Tag and ELISAassays, polyacrylamide gel electrophoresis, mass spectroscopy andenzymatic assays. For enzymatic assays, methods for detecting chorismatesynthase and chorismate mutase activity are described above. Alternativemethods have been described in the literature. Also, any reporter genesystem may be used to detect chorismate synthase or chorismate mutaseprotein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame withchorismate synthase or chorismate mutase, so as to produce a chimericpolypeptide. Methods for using reporter systems are known to thoseskilled in the art. Examples of reporter genes include, but are notlimited to, chloramphenicol acetyltransferase (Gorman et al. (1982) MolCell Biol 2:1104; Prost et al. (1986) Gene 45:107-111), β-galactosidase(Nolan et al. (1988) Proc Natl Acad Sci USA 85:2603-2607), alkalinephosphatase (Berger et al. (1988) Gene 66:10), luciferase (De Wet et al.(1987) Mol Cell Biol 7:725-737), β-glucuronidase (GUS), fluorescentproteins, chromogenic proteins and the like.

Chemicals, compounds or compositions identified by the above methods asmodulators of chorismate synthase or chorismate mutase expression oractivity can then be used to control plant growth. For example,compounds that inhibit plant growth can be applied to a plant orexpressed in a plant, in order to prevent plant growth. Thus, theinvention provides a method for inhibiting plant growth, comprisingcontacting a plant with a compound identified by the methods of theinvention as having herbicidal activity. Alternatively, such compoundsmay be applied to or expressed in a particular plant tissue or organ soas to modulate growth of that tissue or organ.

Herbicides and herbicide candidates identified by the methods of theinvention can be used to control the growth of undesired plants,including both monocots and dicots. Examples of undesired plantsinclude, but are not limited to barnyard grass (Echinochloa crus-galli),crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis),perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa),nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium),velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodiumalbum L.), Brachiara plantaginea, Cassia occidentalis, Ipomoeaaristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setariaspp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and thelike.

Having identified chorismate synthase and chorismate mutase as essentialfor plant growth and development, the invention provides compounds forthe inhibition and modulation of plant growth. As described herein,antisense expression of chorismate synthase or chorismate mutase inplant seedlings results in extremely poor growth and developmentalabnormalities. Accordingly, the invention provides polynucleotides thatspecifically inhibit the expression of chorismate synthase or chorismatemutase.

The polynucleotides of the invention are capable of specificallyinhibiting transcription or translation of a plant chorismate synthaseor chorismate mutase RNA, or decreasing the stability thereof. Suchpolynucleotides include, but are not limited to, antisense molecules,ribozymes, sense molecules, interfering double-stranded RNA (dsRNA) andthe like.

The effect of the expression of such polynucleotides on plant growth anddevelopment will depend upon many factors, including but not limited tothe specificity and activity of the polynucleotide, the level ofexpression of the polynucleotide and the expression pattern of thepromoter driving the expression of a polynucleotide of the invention.For example, systemic expression of such polynucleotides can result inplant death or reduced growth. Inducible expression of suchpolynucleotides could result in plant death or decreased growth at thetime of induction. Similarly, developmentally regulated expression couldresult in a reduction of growth or plant death at a particular stage ofdevelopment.

Tissue specific expression would result in a necrosis or reduced growthof that tissue. In preferred embodiments, the polynucleotides of theinvention are operably linked to a tissue-specific or tissue preferredpromoter. In one embodiment, the polynucleotides of the invention areoperably linked to a male-tissue preferred promoter. Maletissue-preferred expression of a polynucleotide of the invention canresult in male-sterile plants. Female tissue-preferred expression of apolynucleotides of the invention can result in seedless plants, or inplants having reduced seed size.

While the polynucleotides of the invention are not limited to aparticular mechanism of action, reduction in chorismate synthase orchorismate mutase gene expression can be mediated at the DNA level andat transcriptional, post-transcriptional, or translational levels. Forexample, it is thought that dsRNA suppresses gene expression by both aposttranscriptional process and by DNA methylation. Sharp and Zamore(2000) Science 287:2431-2433. Ribozyrnes specifically bind andcatalytically cleave RNA. Gene specific inhibition of expression inplants by an introduced sense polynucleotide is termed “cosuppression”.

Antisense polynucleotides, when introduced into a plant cell, arethought to specifically bind to their target polynucleotide and inhibitgene expression by interfering with transcription, splicing, transport,translation and/or stability. Reported mechanisms of antisense actioninclude RNase H-mediated cleavage, activation or inhibition of splicing,inhibition of 5′-cap formation, translation arrest and activation ofdouble strand RNases. See Crooke (1999) Biochim Biophys Acta 1489:31-44.

Antisense polynucleotides can be targeted to chromosomal DNA, to aprimary RNA transcript or to a processed mRNA. Preferred target regionsinclude splice sites and translation initiation and termination codons,and other sequences within the open reading frame.

Thus, in another aspect, the invention provides an isolated nucleic acidfor modulating plant growth, comprising: a polynucleotide selected fromthe group consisting of a plant chorismate synthase-specific ribozyme, aplant chorismate synthase-specific antisense molecule, a plantchorismate synthase-specific dsRNA, a plant chorismate synthase-specificsense molecule, a plant chorismate mutase-specific ribozyme, a plantchorismate mutase-specific antisense molecule, a plant chorismatemutase-specific dsRNA and a plant chorismate mutase-specific sensemolecule.

In preferred embodiments, the polynucleotide is a plant chorismatesynthase-specific antisense molecule or a plant chorismatemutase-specific antisense molecule. Thus, the invention provides anantisense molecule specific to all or a part a polynucleotide selectedfrom the group consisting of: a chorismate synthase primary transcript,a chorismate synthase mRNA, a chorismate mutase primary transcript and achorismate mutase mRNA; wherein said chorismate synthase mRNA is notfrom maize, soybean, wheat or rice.

Preferably the antisense molecule is specific for a plant chorismatesynthase or chorismate mutase mRNA. In one embodiment, the antisensemolecule is specific for an Arabidopsis chorismate synthase orchorismate mRNA. The present inventors are the first to provide thesequence of an Arabidopsis chorismate synthase partial cDNA (SEQ IDNO:1). SEQ ID NO:7 is the antisense or complement of SEQ ID NO:1 Thus,in one aspect, the invention provides an isolated polynucleotideselected from the group comprising:

a) a nucleotide sequence consisting essentially of SEQ ID NO:1 or SEQ IDNO:7;

b) a nucleotide sequence consisting essentially of a sequence having atleast 80% sequence identity with SEQ ID NO:1 or SEQ ID NO:7; and

c) a probe comprising SEQ ID NO:1 or SEQ ID NO:7.

The invention further provides vectors comprising these polynucleotidesand related transformed cells and transformed plants.

The sequences of Arabidopsis chorismate mutase-1, chorismate mutase-2and chorismate mutase-3 full length cDNAs are shown in SEQ ID NOs:2, 3,and 4, respectively. The sequence of the Arabidopsis chorismate mutase-1and chorismate mutase-2 genes are shown in SEQ ID NOs:5 and 6,respectively. A preferred antisense polynucleotide that is specific forthe Arabidopsis chorismate synthase mRNA is shown in SEQ ID NO:7.Antisense sequences corresponding to the Arabidopsis full lengthchorismate mutase-1, chorismate mutase-2 and chorismate mutase-3 cDNAsare shown in SEQ ID NOs:8, 9, and 10, respectively.

By “plant chorismate synthase-specific” and “plant chorismatemutase-specific” polynucleotides is meant that the polynucleotide canspecifically hybridize to either the sense or antisense strand of aplant chorismate synthase or chorismate mutase gene or RNA. The termsplant “chorismate synthase RNA” or “chorismate mutase RNA” include boththe primary transcript and the processed mRNA. For example, a portion ofa ribozyme will hybridize with all or a portion of a target primarytranscript or spliced mRNA. Similarly, all or a portion of an antisensemolecule will hybridize with a target primary transcript or splicedmRNA. In contrast, a sense polynucleotide will have partial or completesequence identity with all or a portion of a target gene and primarytranscript or mRNA. Thus, the sense polynucleotide will hybridize to acomplement of a target gene, primary transcript or mRNA. Obviously, adsRNA, when denatured, will hybridize to both a target gene and/or mRNAand the corresponding complement.

By “specifically hybridize” is meant that the polynucleotide willhybridize to the target gene or RNA at a level of at least two-fold overbackground under conditions of high stringency. For example, achorismate synthase-specific antisense polynucleotide will hybridize toa chorismate synthase gene, primary transcript and/or processed mRNA ofat least one plant with a affinity of at least two fold over the levelof hybridization to that plant's other nucleic acids.

The specificity of the hybridization will depend upon many factors,including the length and degree of complementarity between the antisensemolecule and the target sequence, the length of the antisense molecule,the temperature of the hybridizations and washes, and the salt,detergent and formamide concentrations of the hybridization and washbuffers.

In preferred embodiments, the polynucleotides of the invention willhybridize to at least one plant chorismate synthase or chorismate mutasegene or RNA under high stringency hybridization conditions. Thus, in oneembodiment, the invention provides an antisense molecule that hybridizesunder high stringency conditions to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

It is understood that the polynucleotides of the invention need not becompletely complementary to the target chorismate synthase or chorismatemutase gene or RNA, nor that they hybridize to each other along theirentire length, in order to modulate expression or to form specifichybrids. Furthermore, the polynucleotides of the invention need not befull length with respect to the target plant chorismate synthase orchorismate mutase gene or RNA. In general, greater homology cancompensate for shorter polynucleotide length.

Typically the polynucleotides of the invention will comprise anucleotide sequence having 60-100% sequence identity with at least 14,15, 16, 17, 18, 19, 20, 25, or at least 30 consecutive nucleotides of asense or antisense strand of a plant chorismate synthase or chorismatemutase gene or RNA. Preferably, the sequence identity will be at least70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% and mostpreferably at least 99%.

In one embodiment, the invention provides an antisense molecule, whereinsaid molecule comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10 or at least 14 consecutive nucleotides thereof.

In another embodiment, the invention provides an antisense molecule,wherein said molecule has at least 95% sequence identity with at least20, 25, 30, 40 or 50 consecutive nucleotides of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the antisensemolecule has at least 80%, 85%, 90%, 95% 98% 99% or 100% sequenceidentity with at least 100 consecutive nucleotides of SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

The polynucleotides of the invention may be specific for any one or moreplant chorismate synthase of chorismate mutase genes or RNAs. Thegenomic sequence and/or cDNA of chorismate synthases and chorismatemutases from a variety of organisms are known. Examples include, but arenot limited to those described in Braun et al. (1996) Planta 200:64-70(Lycopersicon esculentum chorismate synthase); Charles et al. (1990) JGen Microbiol 136:353-358 (C. sempervirens chorismate synthase); GenBankaccession No:AW568644, AW279504, AW277636 and A1941440 (Glycine max(soybean) chorismate synthase cDNA); GenBank Accession No: A1729656(Cotton fiber Gossypium hirsutum chorismate synthase cDNA); Henstrand etal. (1995) J Biol Chem 270:20447-20452 (Neurospora crassa chorismatesynthase cDNA); Jones et al. (1991) Mol Microbiol 5:2143-2152(Saccharomyces cerevisiae chorismate synthase); Schaller et al. (1991) JBiol Chem 266:21434-21438 (Bacillis subtilis chorismate synthase);Roberts et al. (1998) Nature 393:801-805 (T. gondii and P. falciparumchorismate synthases); Horseburgh et al. Microbiology 142:2943-2950(1996) (Staphlococcus aureus chorismate synthase); Mobley et al. (1999)Gene 240:115-123 (A. thaliana chorismate mutase-1, 2 and 3); Lambert etal. (1999) Mol Plant Microbe Interact 12:328-336 (Meloidogyne javanicachorismate mutase); Eberhard et al. (1996) Plant Mol Biol 4:917-922(tomato cytosolic chorismate mutase); Eberhard et al (1996) Plant J5:815-821 (A. thaliana cytosolic and plastidic chorismate mutase);GenBank accession No:AW666427 (Glycine max chorismate mutase cDNA);GenBank Accession No:AW164012 (Lotus japonicus chorismate mutase cDNA);GenBank Accession No:AJ004916 (Prunus avium chorismate mutase mRNA);GenBank Accession No:AA751486 (Rice chorismate mutase-1 cDNA); GenBankAccession No:AF012867 (Petroselinum crispum (parsley) chorismate mutasemRNA); GenBank Accession No:AF012866 (Petroselinum crispum chorismatemutase mRNA) and Krappmann et al. (1999) J Biol Chem 274:22275-22282(Aspergillus nidulans and cerevisae chorismate mutases). Additionalchorismate synthase and chorismate mutase cDNAs have been reported inthe literature and public databases.

An A. thaliana chorismate synthase partial cDNA is disclosed in SEQ IDNO:1. Chorismate mutase-1, chorismate mutase-2 and chorismate mutase-3full length cDNAs are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4,respectively. The A. thaliana chorismate mutase-1 and chorismatemutase-2 genomic DNAs are shown in SEQ ID NO:5 and SEQ ID NO:6,respectively.

Additional plant chorismate synthase and chorismate mutase cDNAs andgenes may be identified using known chorismate synthase and chorismatemutase cDNAs and genes. For example, the sequences shown in SEQ IDNOs:1-6, as well as the sequences of other known chorismate mutase andchorismate synthase genes, cDNAs and proteins can be used to identifyhomologous plant chorismate mutase and chorismate synthase genes andcDNAs. See, for example, Current Protocols in Molecular Biology Ausubelet al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995.

Similarly, the newly identified sequences may be used to obtainadditional plant chorismate mutase and synthase genes and cDNAs. In onemethod, known chorismate synthase and chorismate mutase sequences areused as probes to identify and clone corresponding genes or cDNAs fromplants and plant libraries. Alternatively, synthetic oligonucleotidescan be prepared that correspond to chorismate mutase or chorismatesynthase genes or cDNAs and used as primers in PCR amplification toobtain whole or partial sequences. If desired, these partial sequencescan then be used as probes to isolate chorismate mutase and chorismatesynthase clones from plant genomic or cDNA libraries. Such partial orfull-length sequences can be used to generate the chorismate synthaseand chorismate antisense and sense polynucleotides, dsRNA and ribozymesof the invention. Methods for inhibiting expression in plants usingantisense constructs are known in the art. See, for example, U.S. Pat.Nos. 5,107,065 and 5,254,800, the contents of which are incorporated byreference.

The active antisense molecules of the invention are single stranded RNAor DNA molecules. By active antisense molecule is meant that theantisense molecule is capable of selectively hybridizing with a plantchorismate synthase or chorismate mutase primary transcript or mRNA.However, it is understood that the term antisense molecules includedouble-stranded DNA molecules that encode an antisense RNA.

Preferably, the antisense polynucleotides of the invention are at least8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000nucleotides or more. Antisense polynucleotides can be selected based oncomplementarity to plant chorismate synthase and chorismate mutase genesor RNAs. The complementarity may be to all or a portion of the gene orRNA. Furthermore, the complementarity need not be exact, so long as theantisense molecule is specific for at least one plant chorismate mutaseor chorismate synthase RNA. In general, the degree of complementaritynecessary or antisense inhibition is related to the length of thehybridizing sequences. Preferably, the complementarity is at least 90%,more preferably 95%, even more preferably at least 98% and mostpreferably 100%. Antisense polynucleotides may be designed to bind toexons, introns, exon-intron boundaries, the promoter and other controlregions, such as the transcription and translational initiation sites.Methods for inhibiting plant gene expression using antisense RNAcorresponding to entire and partial cDNA, 3′ non-coding regions, as wellas relatively short fragments of coding regions are known in the art.See, for example, Sheehy et al. (1988) Proc Natl Acad Sci USA85:8805-8809; Cannon et al. (1990) Plant Mol Biol 15:39-47; and Chng etal. (1989) Proc Natl Acad Sci USA 86:10006-10010. Van der Krol et al.(1988) Biotechniques 6:958-976 describe the use of antisense RNA toinhibit plant genes in a tissue-specific manner.

As an alternative to antisense polynucleotides, ribozymes, sensepolynucleotides or dsRNA may be used to reduce plant chorismate mutaseor chorismate synthase gene expression. A ribozyme, or catalytic RNA cancatalyze the hydrolysis of RNA phosphodiester bonds in trans, and thuscan cleave other RNA molecules. Cleavage of a target RNA can decreasestability of the RNA and prevent translation of a full length proteinencoded by that RNA.

Ribozymes contain a first RNA sequence that is complementary to a targetRNA linked to a second enzymatic RNA sequence that catalytically cleavesthe target RNA. Thus, the ribozyme first binds a target RNA throughcomplementary base-pairing, and then acts enzymatically to cut thetarget RNA. Ribozymes may be designed to bind to exons, introns,exon-intron boundaries and control regions, such as the translationalinitiation sites.

At least six types of naturally-occurring enzymatic RNAs, includinghairpin ribozymes and hammerhead ribozymes, have been described. Thehairpin ribozyme can be assembled in various combinations to catalyze aunimolecular, bimolecular or a trimolecular cleavage/ligation reaction(Berzal-Herranz et al. (1992) Genes & Develop 6:129; Chowrira and Burke(1992) Nucleic Acids Res 20:2835; Komatsu et al. (1993) Nucleic AcidsRes 21:185; Komatsu et al. (1994) J Am Chem Soc 116:3692). Increasingthe length of helix 1 and helix 4 regions do not affect the catalyticactivity of the hairpin ribozyme (Hisamatsu et al., supra; Chowrira andBurke, supra; Anderson et al. (1994) Nucleic Acids Res 22:1096). For areview of various ribozyme motifs, and hairpin ribozyme in particular,see Ahsen and Schroeder (1993) Bioessays 15:299; Cech (1992) Curr OpiStruc Bio 2:605; and Hampel et al. (1993) Methods: A Companion toMethods in Enzymology 5:37.

The invention provides ribozymes that are specific for at least oneplant chorismate synthase RNA or plant chorismate mutase RNA. In oneembodiment, the ribozyme is specific for an Arabidopsis chorismatesynthase or chorismate mutase RNA. A ribozyme that is “specific for atleast one plant chorismate synthase RNA or plant chorismate mutase RNA”will contain a polynucleotide sequence that specifically hybridizes to atarget plant chorismate synthase or chorismate mutase primary transcriptor mRNA (the “target”) and cleaves that target. The portion of theribozyme that hybridizes to the transcript or RNA is typically at least7 nucleotides in length. Preferably, this portion is at least 8, 9, 10,12, 14, 16, 18 or 20 or more nucleotides in length. The portion of theribozyme that hybridizes to the target need not be completelycomplementary to the target, as long as the hybridization is specificfor the target. In preferred embodiments the ribozyme will contain aportion having at least 7 or 8 nucleotides that have 100%complementarity to a portion of the target RNA.

In one embodiment, the target RNA is an Arabidopsis chorismate synthaseor chorismate mutase RNA. Accordingly, the invention provides a ribozymespecific for an Arabidopsis chorismate synthase RNA, wherein saidribozyme comprises at least 7 consecutive nucleotides of SEQ ID NO:7.Alternatively, the invention provides a ribozyme specific for anArabidopsis chorismate mutase RNA, wherein said ribozyme comprises atleast 7 consecutive nucleotides of SEQ ID NO:8, SEQ ID NO:9 or SEQ IDNO:10.

Methods for designing and preparing ribozymes are known to those skilledin the art. See, for example, U.S. Pat. Nos. 6,025,167; 5,773,260;5,695,992; 5,545,729; 5.496,698 and 4,987,071, the contents of which areincorporated by reference; Van Tol et al. (1991) Virology 180:23;Hisamatsu et al. (1993) Nucleic Acids Symp Ser 29:173; Berzal-Herranz etal. (1993) EMBO J 12:2567 (describing essential nucleotides in thehairpin ribozyme); Hampel and Tritz, (1989) Biochemistry 28:4929;Haseloff et al. (1988) Nature 334:585-591, Haseloff and Gerlach (1989)Gene 82:43 (describing sequences required for self-cleavage reactions);and Feldstein et al. (1989) Gene 82:53.

In still yet another aspect, the invention provides double-stranded RNA(dsRNA) that is specific for a plant chorismate mutase or chorismatesynthase gene or RNA. The term dsRNA, as used herein, refers to RNAhybrids comprising two strands of RNA. The dsRNAs of the invention maybe linear or circular in structure. The hybridizing RNAs may besubstantially or completely complementary. By substantiallycomplementary, is meant that when the two hybridizing RNAs are optimallyaligned using the BLAST program as described above, the hybridizingportions are at least 95% complementary. Preferably, the dsRNA will beat least 100 base pairs in length. Typically, the hybridizing RNAs ofwill be of identical length with no over hanging 5′ or 3′ ends and nogaps. However, dsRNAs having 5′ or 3′ overhangs of up to 100 nucleotidesmay be used in the methods of the invention.

Thus, in one embodiment, the invention provides a dsRNA, comprising: afirst ribonucleic acid having at least 80% sequence identity with atleast 100 consecutive nucleotides of a plant chorismate synthase RNA ora plant chorismate mutase RNA; and a second ribonucleic acid that issubstantially complementary to said first ribonucleic acid.

Preferably, the first ribonucleic acid of the dsRNA of the invention hasat least 80% sequence identity with at least 100 consecutive nucleotidesof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6. Alternatively, the first ribonucleic hybridizes to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under high stringencyconditions.

The dsRNA may comprise ribonucleotides or ribonucleotide analogs, suchas 2′-O-methyl ribosyl residues or combinations thereof. See U.S. Pat.Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinicacid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393.

Methods for making and using dsRNA are known in the art. One methodcomprises the simultaneous transcription of two complementary DNAstrands, either in vivo, or in a single in vitro reaction mixture. See,for example, U.S. Pat. No. 5,795,715, the contents of which isincorporated by reference. dsRNA can be introduced into a plant or plantcell directly by standard transformation procedures. Alternatively,dsRNA can be expressed in a plant cell by transcribing two complementaryRNAs.

Other methods for the inhibition of endogenous chorismate mutase andchorismate synthase gene expression, such as triple helix formation(Moser et al. (1987) Science 238:645-650 and Cooney et al. (1988)Science 241:456-459) and cosuppression (Napoli et al. (1990) The PlantCell 2:279-289) are known in the art. Partial and full-length cDNAs havebeen used for the cosuppression of endogenous plant genes. See, forexample, U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020 and 5,283,184,the contents of which are incorporated by reference, Van der Kroll etal. (1990) The Plant Cell 2:291-299, Smith et al. (1990) Mol GenGenetics 224:477-481 and Napoli et al. (1990) The Plant Cell 2:279-289.

For sense suppression, it is believed that introduction of a sensepolynucleotide blocks transcription of the corresponding target gene.The sense polynucleotide will have at least 65% sequence identity withthe target plant chorismate synthase or chorismate mutase gene or RNA.Preferably, the percent identity is at least 80%, 90%, 95% or more. Theintroduced sense polynucleotide need not be full length relative to thetarget gene or transcript. Preferably, the sense polynucleotide willhave at least 65% sequence identity with at least 100 consecutivenucleotides of a primary transcript or mRNA of the target endogenousplant gene. The regions of identity can comprise introns and and/orexons and untranslated regions. The introduced sense polynucleotide maybe present in the plant cell transiently, or may be stably integratedinto a plant chromosome or extrachromosomal replicon.

In another aspect, the invention provides a method for theidentification of a candidate chorismate synthase antisense, dsRNA,sense or ribozyme molecule that inhibits a plant chorismate synthaseactivity, comprising:

a) contacting a plant cell expressing a chorismate synthase protein withsaid antisense, dsRNA, sense or ribozyme molecule; and

b) comparing the growth of said cell from step (a) with the growth ofsaid cell in the absence of said antisense, dsRNA, sense or ribozymemolecule; wherein a decrease in growth in the presence of saidantisense, dsRNA, sense or ribozyme molecule is indicative of themolecule being an inhibitor of chorismate synthase activity.

Similarly, the invention provides a method for the identification of acandidate chorismate mutase antisense, dsRNA, sense or ribozyme moleculethat inhibits a plant chorismate mutase activity, comprising:

a) contacting a plant cell expressing a chorismate mutase protein withsaid antisense, dsRNA, sense or ribozyme molecule; and

b) comparing the growth of said cell from step (a) with the growth ofsaid cell in the absence of said antisense, dsRNA, sense or ribozymemolecule; wherein a decrease in growth in the presence of saidantisense, dsRNA, sense or ribozyme molecule is indicative of themolecule being an inhibitor of chorismate mutase activity.

Preferably, the plant cell is a cell in tissue culture. The plant cellmay be contacted with the antisense, dsRNA, sense or ribozyme moleculeby providing the molecule in the tissue culture medium. Alternatively,the plant cell can be contacted with the molecule by expressing it inthe plant cell. Any method for measuring plant cell growth can be used.Such methods are known to those skilled in the art.

Expression of the polynucleotides of the invention in a plant, plantcell or plant tissue will result in the modulation of plant growth ordevelopment. Accordingly, the invention provides recombinant expressioncassettes, comprising the antisense, sense, dsRNA or ribozymepolynucleotides of the invention, wherein said polynucleotide isoperably linked to a promoter that can be active in a plant cell.

The expression cassettes of the invention contain 5′ and 3′ regulatorysequences necessary for transcription and termination of thepolynucleotide of interest. Thus, the expression cassettes will includea promoter and a transcriptional terminator. Other functional sequencesmay be included in the expression cassettes of the inventions. Suchfunctional sequences include, but are not limited to, introns, enhancersand translational initiation and termination sites and polyadenylationsites. The control sequences can be those that can function in at leastone plant, plant cell or plant tissue. These sequences may be derivedform one or more genes, or can be created using recombinant technology.

Promoters useful in the expression cassettes of the invention includeany promoter that is capable of initiating transcription in a plantcell. Such promoters include, but are not limited to those that can beobtained from plants, plant viruses and bacteria that contain genes thatare expressed in plants, such as Agrobacterium and Rhizobium.

The promoter may be constitutive, inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred ororgan-preferred. Constitutive promoters are active under mostconditions. Examples of constitutive promoters include the CaMV 19S and35 S promoters (Odell et al. (1985) Nature 313:810-812), the 2× CaMV 35Spromoter (Kay et al. (1987) Science 236:1299-1302) the Sepl promoter,the rice actin promoter (McElroy et al. (1990) Plant Cell 2:163-171),the Arabidopsis actin promoter, the ubiquitan promoter (Christensen etal. (1989) Plant Molec Biol 18:675-689); pEmu (Last et al. (1991) TheorAppl Genet 81:581-588), the figwort mosaic virus 35S promoter, the Smaspromoter (Velten et al. (1984) EMBO J 3:2723-2730), the GRP1-8 promoter,the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),promoters from the T-DNA of Agrobacterium, such as mannopine synthase,nopaline synthase, and octopine synthase, the small subunit of ribulosebiphosphate carboxylase (ssuRUBISCO) promoter, and the like.

Inducible promoters are active under certain environmental conditions,such as the presence or absence of a nutrient or metabolite, heat orcold, light, pathogen attack, anaerobic conditions, and the like. Forexample, the hsp80 promoter from Brassica is induced by heat shock, thePPDK promoter is induced by light, the PR-1 promoter from tobacco,Arabidopsis and maize are inducible by infection with a pathogen, andthe Adhl promoter is induced by hypoxia and cold stress.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, and leaf-preferred, stigma-preferred,pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred,pedicel-preferred, silique-preferred, stem-preferred, root-preferredpromoters and the like.

In a preferred embodiment, the promoter is a male tissue-preferredpromoter. Male tissues include pollen, tapetum, anther, tassel, pollenmother cells and microspores. Ms45 is an example of a male-preferredpromoter (U.S. Pat. No. 6,037,523). Other tissue preferred,developmental stage preferred and/or inducible promoters include, butare not limited to Prha (expressed in root, seedling, lateral root,shoot apex, cotyledon, petiol, inflorescence stem, flower, stigma,anthers, and silique, and auxin-inducible in roots); VSP2 (expressed inflower buds, flowers, and leaves, and wound inducible); SUC2 (expressedin vascular tissue of cotyledons, leaves and hypocotyl phloem, flowerbuds, sepals and ovaries); AAP2 (silique-preferred); SUC1 (Anther andpistil preferred); AAP1 (seed preferred); Saur-AC1 (auxin inducible incotyledons, hypocotyl and flower); Enod 40 (expressed in root, stipule,cotyledon, hypocotyl and flower); amd VSP1 (expressed in young siliques,flowers and leaves).

Seed preferred promoters are preferentially expressed during seeddevelopment and/or germination. For example, seed preferred promoterscan be embryo-preferred, endosperm preferred and seed coat-preferred.See Thompson et al. (1989) BioEssays 10:108. Examples of seed preferredpromoters include, but are not limited to cellulose synthase (celA),Ciml, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1) and the like.

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the prolifera promoter, the Ap3promoter, the β-conglycin promoter, the phaseolin promoter, the napinpromoter, the soy bean lectin promoter, the maize 15 kD zein promoter,the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter,the waxy, shrunken 1, shrunken 2 and bronze promoters, the Zm 13promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546) and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). Some examples of such heterologous DNA bindingdomains include the LexA and GAL4 DNA binding domains. The LexADNA-binding domain is part of the repressor protein LexA fromEscherichia coli (E. Coli) (Brent and Ptashne, Cell 43:729-736 (1985)).In one preferred embodiment, the promoter comprises a minimal promoteroperably linked to an upstream activation site comprising fourDNA-binding domains of the yeast transcriptional activator GAL4.Schwechheimer et al. (1998) Plant Mol Biol 36:195-204. The sequence ofthis promoter is shown in SEQ ID NO:11.

Polyadenlation signals include, but are not limited to, theAgrobacterium octopine synthase signal (Gielen et al. (1984) EMBOJ3:835-846) and the nopaline synthase signal (Depicker et al. (1982) Moland Appl Genet 1:561-573).

Transcriptional termination regions include, but are not limited to, theterminators of the A. tumefaciens Ti plasmid octopine synthase andnopaline synthase genes. See Ballas et al. (1989) Nuc Acid Res17:7891-7903, Guerineau et al. (1991) Mol Gen Genet 262:14144, Joshi etal. (1987) Nuc Acid Res 15:9627-9639, Mogen et al. (1990) Plant Cell2:1261272, Munroe et al. (1990) Gene 91:15158, Proudfoot (1991) Cell64:671-674, and Sanfacon et al. (1991) Genes Devel 5:14149. Iftranslation of the transcript is desired, translational start and stopcodons can also be provided.

The expression cassettes of the invention may be covalently liked to apolynucleotide encoding a selectable or screenable marker. Examples ofsuch markers include genes encoding drug or herbicide resistance, suchas hygromycin resistance (hygromycin phosphotransferase (HPT)),spectinomycin (encoded by the aada gene), kanamycin and gentamycinresistance (neomycin phosphotransferase (nptII)), streptomycinresistance (streptomycin phosphotransferase gene (SPT)),phosphinothricin or basta resistance (barnase (bar)), chlorsulfuronreistance (acetolactase synthase (ALS)), chloramphenicol resistance(chloramphenicol acetyl transferase (CAT)), G418 resistance, lincomycinresistance, methotrexate resistance, glyphosate resistance, and thelike. Preferably, the expression cassette is linked to the bar gene. Inaddition, the expression cassettes of the invention may be covalentlylinked to genes encoding enzymes that are easily assayed, for example,luciferase, alkaline phosphatase, β-galactosidase (β-gal),β-glucuronidase (GUS) and the like.

In one embodiment, the invention provides an expression cassette,comprising a polynucleotide encoding a antisense RNA that is specificfor a plant chorismate synthase RNA or plant chorismate mutase RNA,wherein said polynucleotide is operably linked to a promoter that can beactive in a plant cell.

In preferred embodiments, the antisense RNA comprises SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, theantisense RNA has at least 80% sequence identity with at least 20consecutive nucleotides of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQID NO:10. In still another embodiment, the antisense RNA hybridizesunder high stringency conditions to the polynucleotide of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

In another aspect, the invention provides an expression cassette,comprising a polynucleotide encoding a plant chorismatesynthase-specific ribozyme or a plant chorismate mutase-specificribozyme, wherein said polynucleotide is operably linked to a promoterthat can be active in a plant cell. Preferably, the chorismatesynthase-specific ribozyme comprises at least 7, 8, 9, 10, 11, 12, 13,14 or at least 15 consecutive nucleotides of SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 or SEQ ID NO:10.

In still another aspect, the invention provides an expression cassettefor expressing a dsRNA, comprising: a first ribonucleic acid having atleast 80% sequence identity with at least 100 consecutive nucleotides ofa plant chorismate synthase RNA or a plant chorismate mutase RNA; and asecond ribonucleic acid that is substantially complementary to saidfirst ribonucleic acid, wherein each of said first and secondribonucleotides are operably linked to at least one promoter that caninitiate transcription in a plant cell.

In a preferred embodiment, the first ribonucleotide has at least 80%sequence identity with at least 100 consecutive nucleotides of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.Alternatively, the first ribonucleotide hybridizes to SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under high stringency conditions.

In another aspect, the invention provides vectors containing theexpression cassettes of the invention. By “vector” is intended apolynucleotide sequence that is able to replicate in a host cell.Preferably the vector contains genes that serve as markers useful in theidentification and/or selection of transformed cells. Such markersinclude, but are not limited to barnase (bar), G418, hygromycin,kanamycin, bleomycin, gentamicin and the like. The vector can compriseDNA or RNA and can be single or double stranded, and linear or circular.Various plant expression vectors and reporter genes are described inGruber et al. in Methods in Plant Molecular Biology and Biotechnology,Glick et al., eds, CRC Press, pp.89-119, 1993; and Rogers et al. (1987)Meth Enzymol 153:253-277. In a preferred embodiment, the vector is an E.coli/A. tumefaciens binary vector. Most preferably, the expressioncassette is inserted between the right and left borders of a TDNA froman Agrobacterium Ti plasmid.

Introduction of the polynucleotides of the invention (includingexpression cassettes and vectors) into a plant, plant cell or planttissue will result in the modulation of plant growth. Thus, in oneaspect, the invention provides plants, plant cells and plant tissuestransformed with at least one polynucleotide, expression cassette orvector of the invention. By transformation is meant the introduction ofa polynucleotide into a target plant cell or plant tissue.

Antisense polynucleotides, dsRNA and ribozymes can be introduceddirectly into plant cells, in the form of RNA. Alternatively, theantisense polynucleotides, dsRNA and ribozymes of the present inventionmay be provided as RNA via transcription in plant cells transformed withexpression constructs encoding such RNAs.

In a preferred embodiment, a plant or plant cell is transformed with adsRNA, comprising a first strand and a second strand, wherein said firststrand is a polynucleotide having at least 80% sequence identity with atleast 100 consecutive nucleotides of a plant chorismate synthase RNA ora plant chorismate mutase RNA, and said second strand is substantiallycomplementary to said first strand.

Alternatively, a plant or plant cell is transformed with an expressioncassette, comprising a polynucleotide encoding a antisense RNA that isspecific for a plant chorismate synthase RNA or plant chorismate mutaseRNA, wherein said polynucleotide is operably linked to a promoter thatcan be active in a plant cell.

In yet another embodiment, a plant or plant cell is transformed with anexpression cassette, comprising a polynucleotide encoding a plantchorismate synthase-specific ribozyme or a plant chorismatemutase-specific ribozyme, wherein said polynucleotide is operably linkedto a promoter that can be active in a plant cell.

In still another embodiment, a plant or plant cell is transformed withan expression cassette for expressing a dsRNA, comprising: a firstpolynucleotide having at least 80% sequence identity with at least 100consecutive nucleotides of a plant chorismate synthase RNA or a plantchorismate mutase RNA, and a second polynucleotide that is substantiallycomplementary to said first polynucleotide, wherein each of said firstand second polynucleotides are operably linked to at least one promoterthat can initiate transcription in a plant cell.

The polynucleotides of the invention may be introduced into any plant orplant cell. By plants is meant angiospenns (monocotyledons anddicotyledons) and gymnosperms, and the cells, organs and tissuesthereof. Methods for the introduction of polynucleotides into plants andfor generating transgenic plants are known to those skilled in the art.See, for example, Weissbach & Weissbach (1988) Methods for PlantMolecular Biology, Academic Press, N.Y. and Grierson & Corey (1988)Plant Molecular Biology, 2^(nd) Ed., Blackie, London, Miki et al. (1993)Procedures for Introducing foreign DNA into Plants, CRC Press, Inc.pp.67-80. Such methods include, but are not limited to electroporation(Fromm et al. (1985) Proc Natl Acad Sci 82:5824 and Riggs et al. (1986)Proc Natl Acad Sci USA 83:5602-5606), particle bombardment (U.S. Pat.Nos. 4,945,050 and 5,204,253, the contents of which are incorporated byreference, Klein et al. (1987) Nature 327:70-73, McCabe et al. (1988)Biotechnology 6:923-926), microinjection (Crossway (1985) Mol Gen Genet202:179-185 and Crossway et al. (1986) Biotechniques 4:320-334), siliconcarbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter9:415-418), direct gene transfer (Paszkowski et al. EMBO J 3:2717-2722),protoplast fusion (Fraley et al. (1982) Proc Natl Acad Sci USA79:1859-1863), polyethylene glycol precipitation (Paszowski et al.(1984) EMBO J 3:2717-2722 and Krens et al. (1982) Nature 296:72-74),silicon fiber delivery, agroinfection (U.S. Pat. No. 5,188,958,incorporated herein by reference, Freeman et al. (1984) Plant CellPhysiol 25:1353 (liposome mediated DNA uptake), Hinchee et al. (1988)Biotechnology 6:915-921, Horsch et al. (1984) Science 233:496-498,Fraley et al. (1983) Proc Natl Acad Sci USA 80:4803, Hemalsteen et al.(1984) EMBO J 3:3039-3041, Hooykass-Van Sloteren et al. (1984) Nature311:763-764, Grimslcy et al. (1987) Nature 325:1677-1679, Gould et al.(1991) Plant Physiol 95:426-434, Kindle (1990) Proc Natl Acad Sci USA87:1228 (vortexing method), Bechtold et al. (1995) In Gene Transfer toPlants, Potrykus et al. (Eds) Springer-Verlag, New York, N.Y. pp19-23(vacuum infiltration), Schell (1987) Science 237:1176-1183; and PlantMolecular Biology Manual, Gelvin and Schilperoort, eds., Kluwer,Dordrecht, 1994).

Preferably, the polynucleotides of the invention are introduced into aplant cell by agroinfection. In this method, a DNA construct comprisinga polynucleotide of the invention is inserted between the right and leftT-DNA borders in an Agrobacterium tumefaciens vector. The virulenceproteins of the A. tumefaciens host cell will mediate the transfer ofthe inserted DNA into a plant cell infected with the bacterium. As analternative to the A. tumefaciens/Ti plasmid system, Agrobacteriumrhizogenes-mediated transformation may be used. See Lichtenstein andFuller in: Genetic Engineering, Volume 6, Ribgy (ed) Academic Press,London, 1987; Lichtenstein and Draper, in DNA Cloning, Volume 2, Glover(ed) IRI Press, Oxford, 1985.

If one or more plant gametes are transformed, transgenic seeds andplants can be produced directly. For example, a preferred method ofproducing transgenic Arabidopsis seeds and plants involves agroinfectionof the flowers and collection of the transgenic seeds produced from theagroinfected flowers. Alternatively, transformed plant cells can beregenerated into plants by methods known to those skilled in the art.See, for example, Evans et al, Handbook of Plant Cell Cultures, Vol I,MacMollan Publishing Co. New York, 1983; and Vasil, Cell Culture andSomatic Cell Genetics of Plants, Acad Press, Orlando, Vol II, 1986.

Once a transgenic plant has been obtained, it may be used as a parent toproduce progeny plants and plant lines. Conventional plant breedingmethods can be used, including, but not limited to crossing andbackcrossing, self-pollination and vegetative propagation. Techniquesfor breeding plants are known to those skilled in the art. The progenyof a transgenic plant are included with in the scope of the invention,provided that the progeny contain all or part of the transgenicconstruct.

The transformed plants and plant cells of the invention include theprogeny of said plant or plant cell, as long as the progeny plants orplant cells still contain the antisense expression cassette. Progeny maybe generated by both asexual and sexual methods. Progeny of a plantinclude seeds, subsequent generations of the plant and the seedsthereof.

Introduction of the polynucleotides of the invention into a plant, plantcell or plant tissue will result in the modulation of plant growth ordevelopment. In most cases, the modulation will be a decrease orcessation of growth or development of the plant cells or tissues wherethe polynucleotides of the invention are expressed.

The antisense, ribozymes, dsRNA and sense polynucleotides of theinvention may be directly transformed into a plant cell. Alternatively,the expression cassettes or vectors of the invention may be introducedinto a plant cell. Once in the cell, expression of the antisense,ribozymes, dsRNA and sense polynucleotides of the invention may betransient or stable. Stable expression requires that all or a part ofthe polynucleotide, expression cassette or vector is integrated into aplant chromosome or a stable extra-chromosomal replicon.

Thus, in one aspect, the invention provides a method for modulatingplant growth, comprising: introducing into a plant cell at least onepolynucleotide selected from the group consisting of: a chorismatesynthase-specific ribozyme, a chorismate synthase-specific antisensemolecule, a chorismate synthase-specific dsRNA, a chorismatesynthase-specific sense molecule, a chorismate mutase-specific ribozyme,a chorismate mutase-specific antisense molecule, a chorismatemutase-specific dsRNA and a chorismate mutase-specific sense molecule.

In one embodiment, the invention provides a method for modulating thegrowth of a plant, plant cell or plant tissue, comprising: transformingsaid plant, plant cell or plant tissue with a dsRNA, comprising: a firstribonucleic acid having at least 80% sequence identity with at least 100consecutive nucleotides of a plant chorismate synthase RNA or a plantchorismate mutase RNA; and a second ribonucleic acid that issubstantially complementary to said first ribonucleic acid.

In another embodiment, the invention provides a method for modulatingthe growth of a plant, plant cell or plant tissue, comprising:transforming said plant, plant cell or plant tissue with an expressioncassette, comprising a polynucleotide encoding a antisense RNA that isspecific for a plant chorismate synthase RNA or plant chorismate mutaseRNA, wherein said polynucleotide is operably linked to a promoter thatcan be active in a plant cell. In a preferred embodiment, the promotercomprises a minimal promoter operably linked to an upstream activationsite comprising four DNA-binding domains of the yeast transcriptionalactivator GAL4.

In yet another embodiment, the invention provides a method formodulating the growth of a plant, plant cell or plant tissue,comprising: transforming said plant, plant cell or plant tissue with anexpression cassette, comprising a polynucleotide encoding a plantchorismate synthase-specific ribozyme or a plant chorismatemutase-specific ribozyme, wherein said polynucleotide is operably linkedto a promoter that can be active in a plant cell.

In still another embodiment, the invention provides a method formodulating the growth of a plant, plant cell or plant tissue,comprising: transforming said plant, plant cell or plant tissue with anexpression cassette for expressing a dsRNA, comprising: a firstribonucleic acid having at least 80% sequence identity with at least 100consecutive nucleotides of a plant chorismate synthase RNA or a plantchorismate mutase RNA; and a second ribonucleic acid that issubstantially complementary to said first ribonucleic acid, wherein eachof said first and second ribonucleotides are operably linked to at leastone promoter that can initiate transcription in a plant cell.

Male tissue-preferred expression of any of these RNAs in one or moremale tissues will result in a male sterile plant. In general, the plantprogeny obtained by cross-pollination show more vigor than the progenyobtained through self-pollination.

Thus, the invention provides a method for generating a male sterileplant, comprising:

a) transforming a plant cell with an expression cassette comprising apolynucleotide selected from the group consisting of: a plant chorismatesynthase-specific ribozyme, a plant chorismate synthase-specificantisense molecule, a plant chorismate synthase-specific dsRNA, a plantchorismate synthase-specific sense molecule, a plant chorismatemutase-specific ribozyme, a plant chorismate mutase-specific antisensemolecule, a plant chorismate mutase-specific dsRNA and a plantchorismate mutase-specific sense molecule, wherein said polynucleotideis operably linked to a plant male tissue-preferred promoter; and

b) obtaining a plant from said transformed plant cell.

In one embodiment, the male-tissue preferred promoter is apollen-preferred promoter.

Ovule-preferred expression of the polynucleotides, expression cassettesof the invention will result in a reduction of seed size. By “reducedseed size” is meant that the seed is reduced by at least 10%.Preferably, the seed is reduced in size to 25%, 50%, 75%, 90% or isabsent. The seed of any plant may be reduced in size, however preferredplants include cucumbers, tomatoes, melons, cherries, grapes,pomegranates and the like.

Thus, the invention provides a method for generating a plant withreduced seed size, comprising: a) transforming a plant cell with theexpression cassette comprising a polynucleotide selected from the groupconsisting of: a plant chorismate synthase-specific ribozyme, a plantchorismate synthase-specific antisense molecule, a plant chorismatesynthase-specific dsRNA, a plant chorismate synthase-specific sensemolecule, a plant chorismate mutase-specific ribozyme, a plantchorismate mutase-specific antisense molecule, a plant chorismatemutase-1-specific dsRNA and a plant chorismate mutase-specific sensemolecule, wherein said polynucleotide is operably linked to anovule-preferred promoter; and

b) obtaining a plant from said transformed plant cell.

EXPERIMENTAL

Plant Growth Conditions

Unless, otherwise indicated, all plants were grown Scotts Metro-Mix™soil (the Scotts Comapnay) in an environmental growth room at 22° C.,65% humidity, 65% humidity and a light intensity of ˜100 μ-E m⁻² s⁻¹supplied over 16 hour day period.

Seed Sterilization

All seeds were surface sterilized before sowing onto phytagel platesusing the following protocol.

1. Place approximately 20-30 seeds into a labeled 1.5 mL conical screwcap tube. Perform all remaining steps in a sterile hood using steriletechnique.

2. Fill each tube with 1 mL 70% ethanol and place on rotisserie for 5minutes.

3. Carefully remove ethanol from each tube using a sterile plasticdropper; avoid removing any seeds.

4. Fill each tube with 1 mL of 30% Clorox and 0.5% SDS solution andplace on rotisserie for 10 minutes.

5. Carefully remove bleach/SDS solution.

6. Fill each tube with 1 mL sterile dI H₂O; seeds should be stirred upby pipetting of water into tube. Carefully remove water. Repeat 3 to 5times to ensure removal of Clorox/SDS solution.

7. Fill each tube with enough sterile dI H₂O for seed plating (˜200-400uL). Cap tube until ready to begin seed plating.

Plate Growth Assays

Surface sterilized seeds were sown onto plate containing 40 ml halfstrength sterile MS medium (no sucrose) and 1% Phytagel using thefollowing protocol:

1. Using pipette man and 200 uL tip, carefully fill tip with seeds and0.1% agarose solution. Place 10 seeds across the top of the plate, about¼ in down from the top edge of the plate.

2. Place plate lid ¾ of the way over the plate and allow to dry for 30minutes or until agarose solution is dry. It is important to allowagarose solution to dry completely before sealing up plates in order toprevent contamination.

3. Using sterile micropore tape, seal the edge of the plate where thetop and bottom meet.

4. Place plates stored in a vertical rack in the dark at 4° C. for threedays.

5. Three days after sowing, the plates transferred into a growth chamberwith a day and night temperature of 22 and 20° C., respectively, 65%humidity and a light intensity of ˜100 μ-E m⁻² s⁻¹ supplied over 16 hourday period.

6. Beginning on day 3, daily measurements are carried out to track theseedlings development until day 14. Seedlings are harvested on day 14(or when root length reaches 6 cm) for root and rosette analysis.

Example 1 Construction of a Transgenic Plant Expressing the Driver

The “Driver” is an artificial transcription factor comprising a chimeraof the DNA-binding domain of the yeast GAL4 protein (amino acid residues147) fused to two tandem activation domains of herpes simplex virusprotein VP16 (amino acid residues 413-490). Schwechheimer et al. (1998)Plant Mol Biol 36:195-204. This chimeric driver is a transcriptionalactivator specific for promoters having GAL4 binding sites. Expressionof the driver is controlled by two tandem copies of the constitutiveCaMV 35S promoter. A diagram of the driver expression cassette is shownin FIG. 1.

The driver expression cassette was introduced into Arabidopsis thalianaby agroinfection. Transgenic plants that stably expressed the drivertranscription factor were obtained.

Example 2 Construction of Chorismate Synthase and Chorismate Mutase-1Antisense Expression Cassettes in Binary Vectors

Partial cDNAs for Arabidopsis thaliana chorismate synthase or chorismatemutase-1 were ligated into the PacI/AscI sites of the E.coli/Agrobacterium binary vector PGT3.2 in the antisense orientation.This placed transcription of the chorismate mutase-1 or chorismatesynthase antisense RNA under the control of an artificial promoter thatis active only in the presence of the driver transcription factordescribed above. See FIGS. 2 and 3. The artificial promoter containsfour contiguous binding sites for the GAL4 transcriptional activatorupstream of a minimal promoter comprising a TATA box (SEQ ID NO:11).

The ligated DNA was transformed into E.coli. Kanamycin resistant cloneswere selected and purified. DNA was isolated from each clone andcharacterized by PCR and sequence analysis. pPG252 expresses chorismatesynthase antisense RNA. pPG205 expresses chorismate mutase-1 antisenseRNA. The antisense expression cassette and a constitutive bamaseexpression cassette are located between right and left T-DNA borders.Thus, this DNA can be transferred into a recipient plant cell byagroinfection.

Example 3 Transformation of Agrobacterium with Target ExpressionCassettes

The binary vectors from Example 2 (pPG252 and pPG205) were transformedinto Agrobacterium tumefaciens by electroporation. TransformedAgrobacterium colonies were isolated using Basta selection. Tworesistant colonies were purified. DNA was prepared from each clone. Theinsert was amplified by PCR and sequenced to confirm the sequence andorientation. The clones were stored as frozen glycerol stocks.

Example 4 Construction of an Arabidopsis Chorismate Synthase andChorismate Mutase-1 Antisense Target Plants

The chorismate synthase and chorismate mutase-1 target expressioncassettes were introduced into Arabidopsis thaliana wild type plants bythe following method. Five days prior to agroinfection, the primaryinflorescence of Arabidopsis thaliana plants grown in 2.5 inch pots wereclipped in order enhance the emergence of secondary bolts.

At two days prior to agroinfection, 5 ml LB broth (10 g/L Peptone, 5 g/LYeast extract, 5 g/L NaCl, pH 7.0 plus 25 mg/L kanamycin added prior touse) was inoculated with a clonal glycerol stock of Agrobacteriumcarrying pPG252, pPG205 or pPG238. The cultures were incubated overnightat 28° C. at 250 rpm until the cells reached stationary phase. Thefollowing morning, 200 ml LB in a 500 ml flask was inoculated with 500μl of the overnight culture and the cells were grown to stationary phaseby overnight incubation at 28° C. at 250 rpm. The cells were pelleted bycentrifugation at 8000 rpm for 5 minutes. The supernatant was removedand excess media was removed by setting the centrifuge bottles upsidedown on a paper towel for several minutes. The cells were thenresuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and250 μl/L Silwet L-77™ (84% polyalkyleneoxide modifiedheptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether),and transferred to a one liter beaker.

The previously clipped Arabidopsis plants were dipped into theAgrobacterium suspension so that all above ground parts were immersedand agitated gently for 10 seconds. The dipped plants were then coverwith a tall clear plastic dome in order to maintain the humidity, andreturned to the growth room. The following day, the dome was removed andthe plants were grown under normal light conditions until mature seedswere produced. Mature seeds were collected and stored desiccated at 4°C.

Transgenic Arabidopsis T1 seedlings were selected using glufosinatetreatment. Approximately 70 mg seeds from an agrotransformed plant weremixed approximately 4:1 with sand and placed in a 2 ml screw cap cryovial.

The surface of the seeds was sterilized using the chlorine gas method.Briefly, the open vials were placed in a vacuum desiccator in a safetyhood. A glass beaker containing 200 ml 5.25% sodium hypochloritesolution was placed in the desiccator. Two ml concentrated HCl was addedto the hypochlorite solution and the cover was placed on the desiccator.Vacuum was applied briefly to seal the dessicator, and the seeds wereleft in the desiccator overnight.

One vial of sterilized seeds was then sown in a cell of an 8 cell flat.The flat was covered with a dome, stored at 4° C. for 3 days, and thentransferred to a growth room. The domes were removed when the seedlingsfirst emerged. After the emergence of the first primary leaves, the flatwas sprayed uniformly with a 1:3000 dilution of Liberty™ (AgrEvo; 11.3%glufosinate) in water, 0.005% Silwet (50 μl/L) until the leaves werecompletely wetted. The spraying was repeated for the following two days.

Ten days after the first spraying resistant plants were transplanted to2.5 inch round pots containing moistened sterile potting soil. Thetransplants were then sprayed with herbicide and returned to the growthroom. These herbicide resistant plants represent stably transformed T1plants. Mature T1 plants are then dried and harvested for T2 seeds.

Example 5 Effect of Chorismate Synthase Antisense Expression inArabidopsis Seedlings

The chorismate synthase target plants from two transformed plant linesobtained in Example 5 were crossed with the Arabidopsis transgenicdriver line described above. The resulting F1 seeds were then subjectedto a PGI plate assay to observe seedling growth over a 2-week period.Seedlings were inspected daily for growth and development. During thisperiod, a portion of seedlings derived from one of the chorismatesynthase antisense target lines developed very slowly and had varyinglevels of developmental abnormalities. The progeny from the second linewere all normal. FIG. 4 shows the effect of chorismate synthaseantisense expression on Arabidopsis seedlings. The results aresummarized in Table 1.

TABLE 1 Phenotypes of plants expressing chorismate synthase antisenseconstructs Construct No. Wild Type No. Abnormal χ² Value^(a)Probability^(a) PPG252 5 5 0.00 1.000 ^(a)Chi-square and P values (0.05)were obtained to evaluate the hypothesis that chlorosis and wild-typephenotypes are segregating in a 1:1 ratio.

The identification of one line expressing an antisense chorismatesynthase RNA that produces progeny that segregate 1:1 for abnormalphenotypes when crossed with a driver line demonstrates that thechorismate synthase gene is essential for normal plant growth anddevelopment. The fact that the progeny derived from the other line didnot exhibit any abnormal phenotypes is not unexpected, since it is wellknown that antisense expression does not work equally well in allindependently transformed lines containing the same construct. Forexample, the failure of these lines to produce abnormal progeny whencrossed with the driver plant could be due to positional silencing ofantisense expression.

Example 6 Effect of Chorismate Mutase-1 Antisense Expression inArabidopsis Seedlings

The chorismate mutase-1 target plants from five transformed plant linesobtained in Example 5 were crossed with the Arabidopsis transgenicdriver line described above. The resulting F1 seeds were then subjectedto a PGI plate assay to observe seedling growth over a 2-week period.Seedlings were inspected daily for growth and development. FIG. 5 showsthe effect of chorismate mutase antisense expression on Arabidopsisseedlings. A portion of seedlings derived from one of the chorismatesynthase antisense target lines developed very slowly and had varyinglevels of developmental abnormalities. The results are summarized inTable 2.

TABLE 2 Phenotypes of plants expressing chorismate mutase-1 antisenseconstructs Line No. Wild Type No. Abnormal χ² Value^(a) Probability^(a)pPG205 4 5 0.11 0.74 (line 1) pPG205 5 4 0.11 0.74 (line 2) pPG205 3 61.0 0.32 (line 3) ^(a)Chi-square and P values (0.05) were obtained toevaluate the hypothesis that chlorosis and wild-type phenotypes aresegregating in a 1:1 ratio.

The clear 1:1 segregation ratio observed in three out of fiveindependent CM1 antisense lines demonstrates that the antisenseexpression of a chorismate mutase-1 gene results in significantlyimpaired growth. Thus, chorismate mutase-1 represents and essential genefor normal plant growth and development. In addition, these results showthat both cDNA clones and genomic DNA can be used for antisenseconstructs.

Example 7 In vitro Assays for Modulators of Chorismate Synthase Activity

Chorismate synthase activity is determined according to the assay ofMacheroux et al. (1996) J Biol Chem 271:25850-25858. Briefly, chorismatesynthase activity is measured spectrophotometricly in a quartz cuvettecontaining 1 ml 5-10 μM reduced flavin, 90 μM EPSP, 1 mM potassiumoxalate, 50 mM MOPS buffer, 10% glycerol, pH 7.5, in the presence orabsence of a compound to be tested for the ability to modulatechorismate synthase activity. 5 μl of 23 μM chorismate synthase isadded, and the change in absorbance at 275 nm (Δε=2630 M⁻¹ cm⁻¹), 25° C.was measured as a baseline. The absorbance is compared in the presenceand absence of the test compound.

While the foregoing describes certain embodiments of the invention, itwill be understood by those skilled in the art that variations andmodifications may be made and still fall within the scope of theinvention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1 <211> LENGTH: 1053<212> TYPE: DNA <213> ORGANISM: Arabidopsis <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1053)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1caatcccttt gttgagttgg gaatacagtt ttgcagttct ggtcagttta tg#aacaccgt     60ttacaagccc aagtgggatt tccgacaatg caggtcaaaa cccaccaaca ag#atttcctt    120atgctcttga cggattcatt cgcagttatc tctttttcgg attattcgga tt#tagggctt    180ctgacaatgt tattttctat ctgtcggagt gtaaattctc gtgtctacca at#tcttctgg    240aagtacacat tggtgaactt gcgagacata ggcaagattc tcagttcctg ca#aattgctt    300caaaattctc ttgcccaaag ctccaggagc aactcttcca atggtctctc ta#gctgaaga    360tcttcctcca ccctgcactg atctgacacc atacttcatg tcataagttg ca#tcagcatg    420cgatggtcta taggcaaccg acatttcact gtaatcaagt cctctctgat ct#gtgtttgg    480tacaaacaca tggataggtg ttcctgtcgt cattccttca gagactccag ac#gatatccg    540gcaagtatca gtctcttttc taggagttgt gatcctgctc tgaccaggcc tc#cttctatc    600gagatcgaat tgcaaatcag attcagtaag tggaatacga ggaggacaac ca#tcaatgat    660acaaccaact cctcctccat gtgattctcc aaaagttgaa actcgaaaat ga#gtcccata    720tgaacttcca gtagcttgta tctggaagtt cttcctggtt tgggtacgga ga#gagatctg    780aacggcggga gaagagagac gacggagctc cgagggaaga gaagaagaac cg#agtttggt    840ggatccgaga atggatttcg aagtgagaga agacgacgcc atgatcagga tt#ccaaggca    900gaagaaagat gattgagaaa ggtttaaagg agagaaattt gaagttggtg gc#aattttga    960gcggacgcgt gggtcgaccg cgggaattcc gagacccggt acctgcatgg cg#tactcagc   1020 tnncncctgg gcnngagttt tccntagaga agt       #                   #       1053 <210> SEQ ID NO 2 <211> LENGTH: 1207<212> TYPE: DNA <213> ORGANISM: Arabidopsis <400> SEQUENCE: 2gcgtcattgt tgatgagatc gtcttgttgc tcctctgcga ttggtgggtt ct#tcgaccat     60cgacgtgaat tatcaacctc aacacccatt tccactcttc ttcctcttcc at#caaccaaa    120tcttctttct ctgttcgttg ttctcttcct cagccatcaa agccacgctc tg#gaaccagc    180tctgttcacg ccgttatgac actcgctgga tcgttgacag ggaagaaacg ag#tggatgag    240agtgagagtt tgactcttga aggtattaga aactctttga tccgtcaaga gg#acagcatt    300atatttgggc tattggagag agccaagtac tgttacaatg ctgatactta tg#atcctact    360gcttttgaca tggatggttt caatggttct ttggttgagt acatggttaa ag#gcactgag    420aagcttcacg ctaaggttgg taggtttaag agtcctgatg aacatccttt ct#tccctgat    480gatctaccag agcctatgtt gcctcctctt cagtacccaa aggtgttgca tt#ttgctgct    540gattcgataa acataaacaa gaagatatgg aacatgtact tcagagacct tg#ttccaaga    600cttgtgaaga aaggcgatga tggtaactac ggctcaacag ctgtctgtga cg#ctatctgc    660cttcagtgtc tctcaaagag aatccattac ggtaaatttg ttgcagaagc ta#aatttcaa    720gcctcacccg aagcatacga gtccgccatc aaagcacaag ataaggatcg ac#tgatggat    780atgctgacat tcccgactgt ggaagatgcg ataaagaaga gagttgagat ga#aaacccga    840acatacgggc aagaagtgaa agttgggatg gaggagaaag aagaagaaga ag#aagaaggg    900aatgaatctc atgtttacaa aatcagtccg atcttagttg gtgacttata tg#gagattgg    960atcatgcctt taacaaaaga ggttcaagtg gagtacttgc tcagaagact gg#actgaggc   1020aacaacaaaa taaacaatat ggctttggta gtagagtaga aaggtttttg aa#tgttcttt   1080ggtttttttt ttttacttta caatatttct aaacgttgtt acactattat tc#cactgtac   1140aaagcgtgca tggtcagtgg tattgaagaa gggtaattag ccgttactca aa#cggtgtcg   1200 tttatgt                  #                  #                   #        1207 <210> SEQ ID NO 3 <211> LENGTH: 1006<212> TYPE: DNA <213> ORGANISM: Arabidopsis <400> SEQUENCE: 3ctttagcatt gaggaagaag aagaagaaag cttcattttt ccaggggata ca#gttgaagc     60ggcatggcaa gagtcttcga atcggattcg ggttctggtt gttccaatgt ac#tgagtctt    120gacttaatca gagaatcgtt gattaggcaa gaagacacca tcgtcttcag ct#tgatcgag    180agagctaagt ttccactcaa ttctcctgct ttcgaggaat ctcgttgtct ag#attctgga    240agtttctctt ctctcactga gtttttcgtc agagagacag aaatcatcca ag#ctaaggta    300ggaagatatg aatacccgga agagaatcct ttcttccttg agaacattcc tc#actcggtt    360tttcctacgc acaaatatcc atcggctttg caccctaagg ctctatctgt ta#acattaac    420aaacaaatct gggatattta ctttaaagaa ttgcttcctt tgtttgtcaa ac#ctggcgat    480gatggcaact atccatcaac tgctgctagt gatctcgcct gtttacaagc tc#tttcgaga    540aggattcact acggtaaatt tgtagctgag gtcaaattca gagatgctcc ac#aagattac    600gagcctgcga ttcgcgctca ggatagagag gctttgatga agctgttgac gt#ttgagaaa    660gtagaagaaa tggttaagaa gagagtgcag aagaaagcag aaacgtttgg ac#aagaagta    720aaattcaact ctggctatgg cgatgagagt aagaagaagt ataaagtgga tc#cattgctt    780gcctctcgca tctacgggga atggcttatc cctctcacta agctcgttga gg#ttgagtat    840cttctacgtc gtctcgattg aatattattt gtatccaaat ctggccctgt ta#aagtgggc    900cttaagtttt taagtgggcc tgttgatatt tgtcaggata tgatagaata at#tgaatgaa    960 gcaacacagt catcactatt ttaaattttg taagatattt taagga   #               1006 <210> SEQ ID NO 4 <211> LENGTH: 1217<212> TYPE: DNA <213> ORGANISM: Arabidopsis <400> SEQUENCE: 4tctcgaccag ataattttgt aacggattgg atttttttgt gtcactatcc ga#tttttatt     60tttcctaact ccgccgaatc cgttcctctg tccttttctt tctccactct ct#gtctctgt    120ctcttcctct tttgattctc cgatggaggc taagttactc aaacccgcgt tt#tacaattc    180cccaaacctc aatcttacga attcttcaag actcatctcg cgattatcaa tc#tggaacga    240taaatcaaaa gttggactat cttctgggtc tctcttcctc cgtctctccg ca#gcttctcc    300gatccgatac tctagggggc tactaagggt agatgagagt gagtatttga aa#cttgaaag    360cattagacac tctttgattc gtcaagagga cagtattatc tttaatcttc tt#gaacgagc    420tcagtatcgc tacaacgctg atacttatga cgaggatgcc tttactatgg aa#gggtttca    480aggatcttta gttgagttta tggtcagaga aactgaaaag cttcacgcaa ag#gtggacag    540gtacaagagt cctgatgagc atcccttttt cccacaatgc ttgcctgaac ct#atccttcc    600tcctattcaa tacccacagg ttttgcatcg ttgcgccgaa tcgataaaca tc#aacaagaa    660ggtgtggaat atgtatttca aacaccttct ccccagactg gtcaagccag gg#gatgacgg    720taattgtggt tcagctgctc tctgtgacac aatgtgtttg cagatacttt ca#aagagaat    780tcacttgcgt aaatttgttg ctgacgccaa gtttcgtgaa aatcctgctg cc#tatgaaac    840agctatcaaa gaacaagacc ggacacagct gatgcaactt ctaacgtacg aa#acggttga    900agaagtagtc aagaagagag ttgagatcaa agccagaatt tttggtcaag ac#ataacgat    960taacgaccca gaaactgaag ctgatccttc ctacaaaata caacctagct ta#gttgcaaa   1020actctatgga gaaaggatca tgcccctcac aaaggaagtc caaattgagt ac#ttgcttag   1080aagactggat taatgttttc aaagaaggaa gcataacatt tgtattgatc ct#atttttcc   1140gaaaaccata atgttgacat aagctgcgca agctagcaag tcaagttgat tt#tattaaaa   1200 aaccctgacg tagctcg              #                  #                   # 1217 <210> SEQ ID NO 5 <211> LENGTH: 5099<212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(5099)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 5ggatccgctc aactcttttg atccttgagg atgatcacga ccgaatgctt tg#aggagaag     60agtggtttat ttttgttgta ttgacaaaaa cttctacaat aactttgttt tg#ctttgaca    120tttctcacaa tttttatctg tctagattcc aaaagcgatt tttaagtgtc ct#tgcaccca    180ctcaaattct gatgattttc tatccaccta atgtggattt cactttaatt ct#ctttttct    240aattaaaata agctactgta ttttttttcc ttagcttgag gacattttta aa#gtttttta    300ttttgaaatc gtcactttct aaaatatatg atttgtagtt tcaactaaaa gc#ggatgaca    360aaagaaaatt gtagttattt tcccaatttt tttgaatcca tagttagtta at#tatttggg    420taatattaca aagtagtcaa aatttgaatc aaaataaagt aattaactat tg#tcagtttt    480ttatcggtaa atgaacatat gacgtcacaa taaaagacaa aaatatccca ac#aagtcgaa    540ccaaaaacaa acacttgcgg aaaaaaaaac atagatatga aagattttaa ca#aaagtaac    600attttataac gaactttttg aaaattagca tgttatatta aaaataccat tt#cactttaa    660aaaaagtaca atgtgaaata aaataataac atgatatgta ttataatagc gt#gtcaaata    720aaaaacaaag ctgattaata ttaaaaagta acatgaaatc ttaagaaact at#tcattttc    780aaccgtcaat tagtattgta attaaaaagt gtgtaagact caaagaatac ta#ttggatca    840aatggttatg ggcttcgttt acgagagaga cgcgatgggt tgagtccttt gt#tttagcaa    900agaaaatttt ttagaaattt ctaaaaatag cactaaaaca agttttatgg ac#aataatag    960cacacactcc atagacttct aaaaatagca ttatttgact tactatgaag tt#tttgttct   1020aaaagtatta tcactaattt attttttcaa taataccaca atatttatat aa#aacctatt   1080caaccaaaat aaattagaaa taaatttaaa atattatatt tgggttctgt at#ttccaatt   1140tatggtatat atttcaacca tttaaaagaa attttgatca ttttaatatt ac#tttatggt   1200attatatttg ggttccctct ttccaattta tgatatatat tttcactgtt ta#aaagagat   1260ttttcatttt aatattagtt tgtggtatta tatttgggtt ctctatttcc aa#tttagggt   1320atatatttcc actatttaaa aaaaatgtga ttcaatttaa tattagtgtt tg#ctctcatt   1380tttatttggg agattgacac aaatgacact taaaaacttt attatttaat ta#atttctaa   1440agaattaaaa actttatgtg ctatttttgg aagtttaaaa aaaaagtgtt at#ttttggaa   1500taataaagtt tttaatgtca tttgtgtcaa ttccacaaaa attaaaaact tt#tataccaa   1560catacaaacg aagtcatttt gagtagacac ctcatcatgc atgtgttagc tg#actcaaca   1620acctattaat tacacaaaat ttcaaatcga aaactatact atacagattg ta#tgagcata   1680aaaatattaa ttcaagacga agtcattaaa atatcgaaaa ctatactata ca#gattgtat   1740gagcataaaa atattatatt ttcatcacaa aagaatcatc tacagtaaaa ac#gtatatat   1800aatagttttt ataatcatat atatgaaagt tggccaactc tctccatatg at#tgatacat   1860catcacttta acatttgata tatcatcact ttaacattta ccatgtgtcc ac#tgaataat   1920taatgtaagc tcttcaactt ttaattttta gattaatgat gtatttatta tt#taatttta   1980ctaatacata atttattttg ttattacatg ttcttcaata aaattatttt at#tggttttc   2040tcatactcga agaatttttt ctgtaatctt aactcaaatt atgagtgtat ag#tagttatg   2100gattagttaa taatttgaat agtttgcata ttgttatata tctatatagt aa#atcgccga   2160gggtgaagag tatgtaagaa gtttaatcat caaattctta ggtttcgaga at#ctcaatat   2220taccttctat ttaattagtt taagacgaac attgtttagg aattgtgtgt at#aatgcatt   2280gttgaaaatg ttgttaggaa aaaatatttt aatactaaaa ataattactt at#tacataag   2340taaatgatta gataattaaa aattatattt aaattaaacg atacaaaaaa tc#taaaaata   2400ctgcagcgta acgtgggtaa ttacctagtt tgtaataaat aataattcaa ga#cgttagaa   2460aaagagagga taattcactg ctcttctata cactatttgt tcatatttaa at#tctttatt   2520cttttcatat cattggtagt ttatatatat attatgtagt tatttaatcc tt#gtttgatt   2580gtttctatgt atcgaccaaa aaaatataca aagttgaatc ttaaaatatt gt#tttcatat   2640taatattcta aactttggta aagttgtaag ttataaaaca acttgaacat aa#aaataatg   2700attgaaatta atgagagaac acaaaataga aaaaaaaagg tcatgagaac ag#aacagaaa   2760cacttttgtg gctttcgtgg gctaaagacg tgcacgcaga cacaacccta aa#catctctc   2820cctctctcac caactctttc tctttaccca ctgcacctac ccccaaacaa tc#ccttttaa   2880catctctcat tcctctgctc atgattcttt gtctcttcct ctgatttctc aa#tcctctgt   2940tttctccgtc tcctctgttt ttttcacatc aatggaagcg tcattgttga tg#agatcgtc   3000ttgttgctcc tctgcgattg gtgggttctt cgaccatcga cgtgaattat ca#acctcaac   3060acccatttcc actcttcttc ctcttccatc aaccaaatct tctttctctg tt#cgttgttc   3120tcttcctcag ccatcaaagc cacgctctgg aaccagctct gttcacgccg tt#atgacact   3180cgctgggtac gaaagtctca atctttagat tctaattgag aaattgagat ta#cccttttt   3240gttacctttc atgatttgta gattcccatt gtgaaattaa cataaccctt tg#tgattttg   3300tagcttaaat tagaaacctt tatgttcttc ttagatgaat ttgaagcaaa gt#tttgtttt   3360tgtttgttgt tgttgttgat atagatcgtt gacagggaag aaacgagtgg at#gagagtga   3420gagtttgact cttgaaggta ttagaaactc tttgatccgt caagaggaca gc#attatatt   3480tgggctattg gagagagcca agtactgtta caatgctgat acttatgatc ct#actgcttt   3540tgacatggat ggtttcaatg gttctttggt tgagtacatg gttaaaggca ct#gagaagct   3600tcacgctaag gtaacaaaca catgctcttt attaacatac cctcaagatt ga#aacttgac   3660tttgttatgg aacttgatta ggttggtagg tttaagagtc ctgatgaaca tc#ctttcttc   3720cctgatgatc taccagagcc tatgttgcct cctcttcagt acccaaaggt ac#tcaatata   3780catgtttcac atgaaaaaag atcgtctcct ttatgttttc ttgcatctta cc#gatatggt   3840ttcttgatgt tcggtgaagc aatgtgtaat cttgtttgag atgtgttttc aa#cttctgta   3900ctttggtgct gaggattcaa gtttctttct tattgtatag gtgttgcatt tt#gctgctga   3960ttcgataaac ataaacaaga agatatggaa catgtacttc agagaccttg tt#ccaagact   4020tgtgaagaaa ggcgatgatg gtaactacng ctcaacagct gtctgtgacg ct#atctgcct   4080tcaggtttgt tccttttttt ccttttgtta ggtatcagaa acaagcttgg at#atttgttt   4140aaaaacttgt cacctctttt ctaagtcgat taacgtctca tgtagttttt ga#tgtccatt   4200gcagtgtctc tcaaagagaa tccattacgg taaatttgtt gcagaagcta aa#tttcaagc   4260ctcacccgaa gcatacgagt ccgccatcaa agcacaagta tttatctact tc#tctaaagc   4320tctcacatac acacaaaaac tcgaagttta tgcattactt accttttgac at#ggcaacat   4380acgcattgca ggataaggat gcactgatgg atatgctgac attcccgact gt#ggaagatg   4440cgataaagaa gagagttgag atgaaaaccc gaacatacgg gcaagaagtg aa#agttggga   4500tggaggagaa agaagaagaa gaagaagaag ggaatgaatc tcatgtttac aa#aatcagtc   4560cgatcttagt tggtgactta tatggagatt ggatcatgcc tttaacaaaa ga#ggttcaag   4620tggagtactt gctcagaaga ctggactaag gcaacaacaa aataaacaat at#ggctttgg   4680tagtagagta gaaaggtttt tgaatgttct ttggtttttt ttttttactt ta#caatattt   4740ctaaacgttg ttacactatt attccactgt acaaagcgtg catggtcagt gg#tattgaag   4800aagggtaatt agccgttact caaacggtgt cgtttatgta catactctca at#tgtggaaa   4860cctgtatatg agttttagtc gctcttattt gttttggaga tgtatttttt tg#tgtgttag   4920tgcctgtaga atgataattg gctgcttagt gtagtggtca ccactggtta ta#tggagttt   4980gactcgtctc atatggagtc cgactccatc cattgtaaaa gtggaaatgg gt#cactagga   5040gagagccttc tcgtcatcct cctgtgttaa cttacaagga aagagccttc ta#gtcatcc    5099 <210> SEQ ID NO 6 <211> LENGTH: 5176 <212> TYPE: DNA<213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 6tctttgggac cactgtcggc agaggcatct tcaacgatgg cctttccttt at#cgcaatga     60tggcatttgt aggagccacc ttccttttcc actatcttca caataaagtg ac#agatagct    120gggcaatgga atccgaggag gtttccggat attacccttt gttgaaaagt ct#caattgcc    180ctttggtctt ctgagactgt atctttgata tttttggagt agacaagtgt gt#cgtgctcc    240accatgttga cgaagatttt cttcttgtca ttgagtcgta agagactctg ta#tgaactgt    300tcgccagtct ttacggcgag ttctgttagg tcctctattt gaatctttga ct#ccatggcc    360tttgattcag tgggaactac ctttttagag actccaatct ctattacttg cc#ttggtttg    420tgaagcaagc cttgaatcgt ccatactgga atagtacttc tgatcttgag aa#atatatct    480ttctctgtgt tcttgatgca gttagtcctg aatcttttga ctgcatcttt aa#ccttcttg    540ggaaggtatt tgatttcctg gagattattg ctcgggtaga tcgtcttgat ga#gacctgct    600gcgtaagcct ctctaaccat ctgtgggtta gcattctttc tgaaattgaa aa#ggctaatc    660tggggacctg caggcatgca agcttcttgg tggaaagaca ctgcttatat ag#tctctgaa    720cgtattccag aaccttctag aatcactcac agctctcgaa ctcatcctta cc#ctctctca    780aggtgagaat ttggtgtttc atatttcatt acaaaatgta aaaaacagag ta#tgtaactt    840ttgtgaaaat gcaacatgtt tattaatgga ttttctcaat tcattccaat gt#aataagta    900gagagatatg agtaccaatt tggtcatgac atcataaaag catagagctt ag#taacttct    960tccccagtag ctccacttct tgggtggctt tcctgatgca aagcatagag tc#tcttctcc   1020tagttcaggc tgctcaagag gagtacatag agtcttagct cctcctcctc ct#cctcctgt   1080ttcatcacct ctggtacgcc tcttcacatc cttctccact tcaaccttat ca#caccatgg   1140agctaaaatc agtttctttt ggctcaaagc ttccacaaat tcatcccaag tt#tcaacctt   1200ttgagtacat tcttcaacct ttctctttgc cacatcatag aggttactct gg#attttctc   1260tagcaagtcc ttaacttgtt caatcaaatc ccctctcttg acatccatct ta#gctccatt   1320gtcacgcgtc actatcctcc ctgatcattg gccaaatctc taggtccagt tt#caattctc   1380aaaggaacac cgttagttct tgatccgcat acttccatcc gcaagaatag tt#gtcgcgta   1440tatctgcttc agcacggatt ccagccccaa gcaaggtgga ttcaacagct tc#acaagcat   1500cacaaagttc ttgataatca gcagcgcctt tgatgggaac atggattaca ac#aacttgaa   1560caggtgccac tttaggaggg aatactaaac ctttgtcatc tccatgagtc at#acacgcgg   1620ctgtagatgt tcaaacatca agcacctcct catcagcctc ttctttggta gc#aaaatcag   1680tgtgtccttc ttgccaaagg aactcacggc tcctgataaa tggggtaggg tc#gctaaact   1740cccatctaac aacgtaacat tagcccactg gttaaccttc aaagggaggt cc#ctgtgtcc   1800tcttatccac cagctcttgt aacccaagca acctctggag caaacccttg ag#tgtggtct   1860ttctccttct caagagaatc acgtgttaca aacatcggga aatagtattc at#ccactttc   1920atcttctcaa tctcagcgtt gaagaacgtt cgtataacgt tccaaatatt ca#tcgctgat   1980gcctgatggc ttgagaatgt agcatccctt tacagactcg tagtattcaa cc#aattcacc   2040aaatctacaa gcctcggaat accacttccc aaaatcttca tctttctttg ca#gtgattcc   2100aagacgagtt tctttcacca tctctttcac tttcctagag gcttctcgag ga#attgcttg   2160acttgtcaca caaccttaga gcgtaagcca gaaaaataat tcagaacaat ac#aaataaac   2220cccagctatt gtgagaagat ataataattc tttatgggcc agtcggtgaa ta#tagttggg   2280cccaaatcat cggatgtgga tcttgttttg atttcaacgc tttcgttttt tt#ttgtttct   2340gttttccttg attgaggaag caatctatgc aaagaggtcc aaaacacttt ag#cattgagg   2400aagaagaaga agaaagcttc atttttccag gtaataagtc ctttgacttt cg#tcattcac   2460aattgctcat ctctatagtt tctatgaatg aattgtttgc ttacttatac ac#catcaagc   2520aggggataca gttgaagcgg catggcaaga gtcttcgaat cggattcggg tt#ctggttgt   2580tccaatgtac tgagtcttga cttaatcaga gaatcgttga ttaggcaaga ag#acaccatc   2640gtcttcagct tgatcgagag agctaagttt ccactcaatt ctcctgcttt cg#aggaatct   2700cgttgtctag attctggaag tttctcttct ctcactgagt ttttcgtcag ag#agacagaa   2760atcatccaag ctaaggtttg cttcccattt taaaaactga tccttttgct aa#aattagat   2820acagagatat caatgcttcg tttgattcgg ttttggtata gcattgtttt ag#attgttcc   2880atgaaattag cagaaagtaa gctacaagtc aacttgattg aggttttaat aa#gcctggat   2940tcttgaatta gcatgccttt tgtttgctat gtgtctcctc cattgcaaaa ga#tgataact   3000tggctttgcc tgtataatct cattgtgtga taacttcttg ttttgatttg ag#tgcgaatc   3060tgccaataaa aggctccgac tttatcatat gtatacgaga tttccttatg aa#aacctcat   3120tatatgtgga gattggaaat ggaggactat tgttttctat ttttataatg tc#tgaaagtc   3180ttatttcatt aatatattca tctcattggt ttatattctt aagtttctgg at#attgagcc   3240tatatgtttg ttcattggtt tacttgaaaa ccttatgtgt atgtgtatat ta#tataggta   3300ggaagatatg aatacccgga agagaatcct ttcttccttg agaacattcc tc#actcggtt   3360tttcctacgc acaaatatcc atcggtatgt atgtagtaaa gtcttgagca tt#tttcttag   3420aactctgaat gctttagtct aacagtactt ttctttctct tgattaggct tt#gcacccta   3480aggctctatc tgttaacatt aacaaacaaa tctgggatat ttactttaaa ga#attgcttc   3540ctttgtttgt caaacctggc gatgatggca actatccatc aactgctgct ag#tgatctcg   3600cctgtttaca agtaaggaga tgattgagta tacataacaa aatcagctct ac#ttttggct   3660aatgatgtct gatctgatat gtttgatctt gtgtaaggct ctttcgagaa gg#attcacta   3720cggtaaattt gtagctgagg tcaaattcag agatgctcca caagattacg ag#cctgcgat   3780tcgcgctcag gtaaacttag tgtcacattg tggattctgt ttcactgtgg tt#ttaaaatg   3840atatgattca caccataatc gtttgatttt cgactgtagg atagagaggc tt#tgatgaag   3900ctgttgacgt ttgagaaagt agaagaaatg gttaagaaga gagtgcagaa ga#aagcagaa   3960acgtttggac aagaagtaaa attcaactct ggctatggcg atgagagtaa ga#agaagtat   4020aaagtggatc cattgcttgc ctctcgcatc tacggggaat ggcttatccc tc#tcactaag   4080ctcgttgagg ttgagtatct tctacgtcgt ctcgattgaa tattatttgt at#ccaaatct   4140ggccctgtta aagtgggcct taagtttata agtgggcctg ttgatatttg tc#aggatatg   4200atagaataat tgaatgaagc aacacagtca tcactatttt aaattttgta ag#atatttta   4260aggaaaagaa aaaagtcgcc agtatttcgc tatcgaaaat cgtttattta ta#tatttgat   4320gattatccat tagagaaccc ttcaaaaaac tctccactca actctctctg gt#cagctgtc   4380tcttccccat tctctagggt tttcaagctc aacctcaagc tccactacga tc#tcttcttc   4440ttctctaatc tcaggtctga atctctcctt cttcactatc tctgatgctt tt#tactgaat   4500ctgattgagg aaactttcca ttttaggatt tgttgatcaa atacactcgg tt#taagaatt   4560aggatcattc tcttttcgat ctagtttcat tagactcgtt cttttagctc tt#gattttat   4620agatctcgtt ttgaggaact gattatttgg ttgttgacag ttgaaagatg ca#aggtgtga   4680ttcgatcctt cgtctccggt ggaaatgttg tgaaaggctc tgtgctgcaa ca#tctccgtg   4740tgattaaccc ggcgattcag ccttctgtgt tttgttcacg ctctgaatca ac#tcaacctg   4800cacgtatgga ggaatctgga ttcgagagca caactatttc cgatgtcatg aa#atccaaag   4860gcaaaagtgc tgatggatct tggctttggt gtactactga tgacactgtt ta#tgatgctg   4920ttaaatccgt atgcttatac tactctcttt ttcctttttt agatatctcg at#gtggattt   4980ggaattgatt gtgtttgatt ttgtttagat gacacaacac aatgttggtg cc#ttggtggt   5040tgtgaaacct ggtgagcaac aagctcttgc tggtatcatt acagagagag gt#aaaattag   5100atctaatcat taataatttt tttgttgtgt cttgtggtat gtgtgattca ct#tttcggca   5160 ttgtgatttc tctaga              #                  #                   #  5176 <210> SEQ ID NO 7 <211> LENGTH: 1053<212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1053)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 7acttctctan ggaaaactcn ngcccaggng nnagctgagt acgccatgca gg#taccgggt     60ctcggaattc ccgcggtcga cccacgcgtc cgctcaaaat tgccaccaac tt#caaatttc    120tctcctttaa acctttctca atcatctttc ttctgccttg gaatcctgat ca#tggcgtcg    180tcttctctca cttcgaaatc cattctcgga tccaccaaac tcggttcttc tt#ctcttccc    240tcggagctcc gtcgtctctc ttctcccgcc gttcagatct ctctccgtac cc#aaaccagg    300aagaacttcc agatacaagc tactggaagt tcatatggga ctcattttcg ag#tttcaact    360tttggagaat cacatggagg aggagttggt tgtatcattg atggttgtcc tc#ctcgtatt    420ccacttactg aatctgattt gcaattcgat ctcgatagaa ggaggcctgg tc#agagcagg    480atcacaactc ctagaaaaga gactgatact tgccggatat cgtctggagt ct#ctgaagga    540atgacgacag gaacacctat ccatgtgttt gtaccaaaca cagatcagag ag#gacttgat    600tacagtgaaa tgtcggttgc ctatagacca tcgcatgctg atgcaactta tg#acatgaag    660tatggtgtca gatcagtgca gggtggagga agatcttcag ctagagagac ca#ttggaaga    720gttgctcctg gagctttggg caagagaatt ttgaagcaat ttgcaggaac tg#agaatctt    780gcctatgtct cgcaagttca ccaatgtgta cttccagaag aattggtaga ca#cgagaatt    840tacactccga cagatagaaa ataacattgt cagaagccct aaatccgaat aa#tccgaaaa    900agagataact gcgaatgaat ccgtcaagag cataaggaaa tcttgttggt gg#gttttgac    960ctgcattgtc ggaaatccca cttgggcttg taaacggtgt tcataaactg ac#cagaactg   1020 caaaactgta ttcccaactc aacaaaggga ttg       #                   #       1053 <210> SEQ ID NO 8 <211> LENGTH: 1207<212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 8acataaacga caccgtttga gtaacggcta attacccttc ttcaatacca ct#gaccatgc     60acgctttgta cagtggaata atagtgtaac aacgtttaga aatattgtaa ag#taaaaaaa    120aaaaaccaaa gaacattcaa aaacctttct actctactac caaagccata tt#gtttattt    180tgttgttgcc tcagtccagt cttctgagca agtactccac ttgaacctct tt#tgttaaag    240gcatgatcca atctccatat aagtcaccaa ctaagatcgg actgattttg ta#aacatgag    300attcattccc ttcttcttct tcttcttctt tctcctccat cccaactttc ac#ttcttgcc    360cgtatgttcg ggttttcatc tcaactctct tctttatcgc atcttccaca gt#cgggaatg    420tcagcatatc catcagtcga tccttatctt gtgctttgat ggcggactcg ta#tgcttcgg    480gtgaggcttg aaatttagct tctgcaacaa atttaccgta atggattctc tt#tgagagac    540actgaaggca gatagcgtca cagacagctg ttgagccgta gttaccatca tc#gcctttct    600tcacaagtct tggaacaagg tctctgaagt acatgttcca tatcttcttg tt#tatgttta    660tcgaatcagc agcaaaatgc aacacctttg ggtactgaag aggaggcaac at#aggctctg    720gtagatcatc agggaagaaa ggatgttcat caggactctt aaacctacca ac#cttagcgt    780gaagcttctc agtgccttta accatgtact caaccaaaga accattgaaa cc#atccatgt    840caaaagcagt aggatcataa gtatcagcat tgtaacagta cttggctctc tc#caatagcc    900caaatataat gctgtcctct tgacggatca aagagtttct aataccttca ag#agtcaaac    960tctcactctc atccactcgt ttcttccctg tcaacgatcc agcgagtgtc at#aacggcgt   1020gaacagagct ggttccagag cgtggctttg atggctgagg aagagaacaa cg#aacagaga   1080aagaagattt ggttgatgga agaggaagaa gagtggaaat gggtgttgag gt#tgataatt   1140cacgtcgatg gtcgaagaac ccaccaatcg cagaggagca acaagacgat ct#catcaaca   1200 atgacgc                  #                  #                   #        1207 <210> SEQ ID NO 9 <211> LENGTH: 1006<212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 9tccttaaaat atcttacaaa atttaaaata gtgatgactg tgttgcttca tt#caattatt     60ctatcatatc ctgacaaata tcaacaggcc cacttaaaaa cttaaggccc ac#tttaacag    120ggccagattt ggatacaaat aatattcaat cgagacgacg tagaagatac tc#aacctcaa    180cgagcttagt gagagggata agccattccc cgtagatgcg agaggcaagc aa#tggatcca    240ctttatactt cttcttactc tcatcgccat agccagagtt gaattttact tc#ttgtccaa    300acgtttctgc tttcttctgc actctcttct taaccatttc ttctactttc tc#aaacgtca    360acagcttcat caaagcctct ctatcctgag cgcgaatcgc aggctcgtaa tc#ttgtggag    420catctctgaa tttgacctca gctacaaatt taccgtagtg aatccttctc ga#aagagctt    480gtaaacaggc gagatcacta gcagcagttg atggatagtt gccatcatcg cc#aggtttga    540caaacaaagg aagcaattct ttaaagtaaa tatcccagat ttgtttgtta at#gttaacag    600atagagcctt agggtgcaaa gccgatggat atttgtgcgt aggaaaaacc ga#gtgaggaa    660tgttctcaag gaagaaagga ttctcttccg ggtattcata tcttcctacc tt#agcttgga    720tgatttctgt ctctctgacg aaaaactcag tgagagaaga gaaacttcca ga#atctagac    780aacgagattc ctcgaaagca ggagaattga gtggaaactt agctctctcg at#caagctga    840agacgatggt gtcttcttgc ctaatcaacg attctctgat taagtcaaga ct#cagtacat    900tggaacaacc agaacccgaa tccgattcga agactcttgc catgccgctt ca#actgtatc    960 ccctggaaaa atgaagcttt cttcttcttc ttcctcaatg ctaaag   #               1006 <210> SEQ ID NO 10 <211> LENGTH: 1217<212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 10cgagctacgt cagggttttt taataaaatc aacttgactt gctagcttgc gc#agcttatg     60tcaacattat ggttttcgga aaaataggat caatacaaat gttatgcttc ct#tctttgaa    120aacattaatc cagtcttcta agcaagtact caatttggac ttcctttgtg ag#gggcatga    180tcctttctcc atagagtttt gcaactaagc taggttgtat tttgtaggaa gg#atcagctt    240cagtttctgg gtcgttaatc gttatgtctt gaccaaaaat tctggctttg at#ctcaactc    300tcttcttgac tacttcttca accgtttcgt acgttagaag ttgcatcagc tg#tgtccggt    360cttgttcttt gatagctgtt tcataggcag caggattttc acgaaacttg gc#gtcagcaa    420caaatttacg caagtgaatt ctctttgaaa gtatctgcaa acacattgtg tc#acagagag    480cagctgaacc acaattaccg tcatcccctg gcttgaccag tctggggaga ag#gtgtttga    540aatacatatt ccacaccttc ttgttgatgt ttatcgattc ggcgcaacga tg#caaaacct    600gtgggtattg aataggagga aggataggtt caggcaagca ttgtgggaaa aa#gggatgct    660catcaggact cttgtacctg tccacctttg cgtgaagctt ttcagtttct ct#gaccataa    720actcaactaa agatccttga aacccttcca tagtaaaggc atcctcgtca ta#agtatcag    780cgttgtagcg atactgagct cgttcaagaa gattaaagat aatactgtcc tc#ttgacgaa    840tcaaagagtg tctaatgctt tcaagtttca aatactcact ctcatctacc ct#tagtagcc    900ccctagagta tcggatcgga gaagctgcgg agagacggag gaagagagac cc#agaagata    960gtccaacttt tgatttatcg ttccagattg ataatcgcga gatgagtctt ga#agaattcg   1020taagattgag gtttggggaa ttgtaaaacg cgggtttgag taacttagcc tc#catcggag   1080aatcaaaaga ggaagagaca gagacagaga gtggagaaag aaaaggacag ag#gaacggat   1140tcggcggagt taggaaaaat aaaaatcgga tagtgacaca aaaaaatcca at#ccgttaca   1200 aaattatctg gtcgaga              #                  #                   # 1217 <210> SEQ ID NO 11 <211> LENGTH: 198<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Artificial promoter comprisi #ng a multiple      cloning site (PacI, AscI, SwaI),  #TATTA box and 4X Gal4      upstream activation site. <400> SEQUENCE: 11ttaattaatc cgggcgcgcc tgatttaaat tggagctcca tggtttaaac ta#ttgatcct     60tcaaatggga atgaacccct ccttatatag aggactgcag ggggatctcg ga#ggagagtc    120ttccggatct cggaggagag tcttccggat ctcggaggag agtcttccgg at#ctcggagg    180 agagtcttcc ggatcccc              #                  #                   # 198

What is claimed is:
 1. A method for identifying a compound as acandidate for a herbicide, comprising: a) combining chorismate and aplant chorismate mutase under reaction conditions suitable forchorismate mutase activity; b) combining chorismate, the plantchorismate mutase and a test compound under the reaction conditions ofstep (a); c) detecting the amount of chorismate and/or prephenate insteps (a) and (b), wherein a difference in the amount detected betweensteps (a) and (b) indicates the test compound as a candidate for aherbicide.
 2. The method of claim 1 wherein the plant chorismate mutaseis from a dicotyledonous plant.
 3. The method of claim 1, wherein theplant chorismate mutase is from a monocotyledonous plant.
 4. The methodof claim 1, wherein the plant chorismate mutase is cytosolic.
 5. Themethod of claim 1, wherein the plant chorismate mutase is plastidic.